MN gene and protein

ABSTRACT

Herein disclosed is a novel oncogene named MN or alternatively MN/CA IX. Abnormal expression of the MN gene is shown to signify oncogenesis, and diagnostic/prognostic methods for pre-neoplastic/neoplastic disease to detect or detect and quantitate such abnormal MN gene expression. Also disclosed are methods to treat pre-neoplastic/neoplastic disease involving the MN gene and protein, e.g., methods comprising the use of MN-specific antibodies, anti-idiotype antibodies thereto, and anti-anti-idiotype antibodies, and the use of MN antisense nucleic acids. Further disclosed are methods to identify and block MN binding site(s) and identify MN protein partners(s).

This application is a divisional of allowed U.S. Ser. No. 09/967,237,filed Sep. 27, 2001, which is a divisional of U.S. Ser. No. 09/178,115,filed Oct. 23, 1998 (now U.S. Pat. No. 6,297,041), which is acontinuation-in-part of U.S. Ser. No. 08/787,739, filed Jan. 24, 1997(now U.S. Pat. No. 6,027,887), which is a continuation-in-part of thefollowing seven U.S. Serial Nos., all of which were filed on Jun. 7,1995: Ser. No. 08/485,049 (now U.S. Pat. No. 6,204,370), Ser. No.08/486,756 (now U.S. Pat. No. 5,981,711), Ser. No. 08/477,504 (now U.S.Pat. No. 5,972,353), Ser. No. 08/481,658 (now U.S. Pat. No. 5,955,075),Ser. No. 08/485,862 (now U.S. Pat. No. 5,989,838), Ser. No. 08/485,863(now U.S. Pat. No. 6,093,548) and Ser. No. 08/487,077 (now U.S. Pat. No.6,069,242). Those seven applications are continuations-in-parts of U.S.Ser. No. 08/260,190, filed Jun. 15, 1994 (now U.S. Pat. No. 6,774,117),which is a continuation-in-part of U.S. Ser. No. 08/177,093, filed Dec.30, 1993 (now U.S. Pat. No. 6,051,226), which is a continuation-in-partof U.S. Ser. No. 07/964,589, filed Oct. 21, 1992 (now U.S. Pat. No.5,387,676). This application declares priority under 35 USC § 120 fromthose U.S. applications and patents.

FIELD OF THE INVENTION

The present invention is in the general area of medical genetics and inthe fields of biochemical engineering, immunochemistry and oncology.More specifically, it relates to the MN gene—a cellular gene consideredto be an oncogene, which encodes the oncoprotein now known alternativelyas the MN protein, the MN/CA IX isoenzyme or the MN/G250 protein.

SEQUENCE LISTING

The Sequence Listing contained on the accompanying two compact discsidentified as “D-0021.5B-2A SEQ LISTING-COPY 1” and “D-0021.5B-2A SEQLISTING-COPY 2” is hereby incorporated by reference. Each SequenceListing was created on May 31, 2006 and is 73.3 kb in size.

BACKGROUND OF THE INVENTION

Zavada et al., International Publication Number WO 93/18152 (published16 Sep. 1993) and U.S. Pat. No. 5,387,676 (issued Feb. 7, 1996),describe the elucidation of the biological and molecular nature of MaTuwhich resulted in the discovery of the MN gene and protein. The MN genewas found to be present in the chromosomal DNA of all vertebratestested, and its expression to be strongly correlated withtumorigenicity.

The MN protein is now considered to be the first tumor-associatedcarbonic anhydrase (CA) isoenzyme that has been described. Carbonicanhydrases (CAs) form a large family of genes encoding zincmetalloenzymes of great physiological importance. As catalysts ofreversible hydration of carbon dioxide, these enzymes participate in avariety of biological processes, including respiration, calcification,acid-base balance, bone resorption, formation of aqueous humor,cerebrospinal fluid, saliva and gastric acid [reviewed in Dodgson etal., The Carbonic Anhydrases, Plenum Press, New York-London, pp. 398(1991)]. CAs are widely distributed in different living organisms.

In mammals, at least seven isoenzymes (CA I-VII) and a few CA-relatedproteins (CARP/CA VIII, RPTP-β, RPTP-T) had been identified[Hewett-Emmett and Tashian, Mol. Phyl. Evol. 5: 50-77 (1996)], whenanalysis of the MN deduced amino acid sequence revealed a strikinghomology between the central part of the MN protein and carbonicanhydrases, with the conserved zinc-binding site as well as the enzyme'sactive center. Then MN protein was found to bind zinc and to have CAactivity. Based on that data, the MN protein is now considered to be theninth carbonic anhydrase isoenzyme—MN/CA IX. [Opavsky et al., Genomics,33: 480-487 (May 1996)]. [See also, Hewett-Emmett, supra, wherein CA IXis suggested as a nomenclatural designation.]

CAs and CA-related proteins show extensive diversity in both theirtissue distribution and in their putative or established biologicalfunctions [Tashian, R. E., Adv. in Genetics, 30: 321-356 (1992)]. Someof the CAs are expressed in almost all tissues (CA II), while theexpression of others appears to be more restricted (CA VI and CA VII insalivary glands). In cells, they may reside in the cytoplasm (CA I, CAII, CA III, and CA VII), in mitochondria (CA V), in secretory granules(CA VI), or they may associate with membrane (CA IV). Occasionally,nuclear localization of some isoenzymes has been noted [Parkkila et al.,Gut, 35: 646-650 (1994); Parkkilla et al., Histochem. J., 27: 133-138(1995); Mori et al., Gastroenterol., 105: 820-826 (1993)].

The CAs and CA-related proteins also differ in kinetic properties andsusceptibility to inhibitors [Sly and Hu, Annu. Rev. Biochem. 64:375-401 (1995)]. In the alimentary tract, carbonic anhydrase activity isinvolved in many important functions, such as saliva secretion,production of gastric acid, pancreatic juice and bile, intestinal waterand ion transport, fatty acid uptake and biogenesis in the liver. Atleast seven CA isoenzymes have been demonstrated in different regions ofthe alimentary tract. However, biochemical, histochemical andimmunocytochemical studies have revealed a considerable heterogeneity intheir levels and distribution [Swensen, E. R., “Distribution andfunctions of carbonic anhydrase in the gastrointestinal tract,” In: TheCarbonic Anhydrases, Cellular Physiology and Molecular Genetics,(Dodgson et al. eds.) Plenum Press, New York, pages 265-287 (1991); andParkkila and Parkkila, Scan J. Gastroenterol., 31: 305-317 (1996)].While CA II is found along the entire alimentary canal, CA IV is linkedto the lower gastrointestinal tract, CA I, III and V are present in onlya few tissues, and the expression of CA VI and VII is restricted tosalivary glands [Parkkila et al., Gut, 35: 646-650 (1994); Fleming etal., J. Clin. Invest., 96: 2907-2913 (1995); Parkkila et al.,Hepatology. 24: 104 (1996)].

MN/CA IX has a number of properties that distinguish it from other knownCA isoenzymes and evince its relevance to oncogenesis. Those propertiesinclude its density dependent expression in cell culture, (e.g., HeLacells), its correlation with the tumorigenic phenotype of somatic cellhybrids between HeLa and normal human fibroblasts, its close associationwith several human carcinomas and its absence from corresponding normaltissues [e.g., Zavada et al., Int. J. Cancer. 54: 268-274 (1993);Pastorekova et al., Virology, 187: 620-626 (1992); Liao et al., Am. J.Pathol., 145: 598-609 (1994); Pastorek et al., Oncogene, 9: 2788-2888(1994); Cote, Women's Health Weekly: News Section, p. 7 (Mar. 30, 1998);Liao et al., Cancer Res., 57: 2827 (1997); Vermylen et al., “Expressionof the MN antigen as a biomarker of lung carcinoma and associatedprecancerous conditions,” Proceedings AACR, 39: 334 (1998); McKiernan etal., Cancer Res. 57: 2362 (1997); and Turner et al., Hum. Pathol.,28(6): 740 (1997)]. In addition, the in vitro transformation potentialof MN/CA IX cDNA has been demonstrated in NIH 3T3 fibroblasts [Pastoreket al., id.].

The MN protein has also recently been identified with the G250 antigen.Uemura et al., “Expression of Tumor-Associated Antigen MN/G250 inUrologic Carcinoma: Potential Therapeutic Target,” J. Urol., 157 (4Suppl.): 377 (Abstract 1475; 1997) states: “Sequence analysis anddatabase searching revealed that G250 antigen is identicial to MN, ahuman tumor-associated antigen identified in cervical carcinoma(Pastorek et al., 1994).”

The MN protein was first identified in HeLa cells, derived from a humancarcinoma of cervix uteri. As indicated above, MN gene expression isstrongly associated with tumorigenicity. It is found in many types ofhuman carcinomas (notably uterine cervical, ovarian, endometrial, renal,bladder, breast, colorectal, lung, esophageal, and prostate, amongothers). Very few normal tissues have been found to express MN proteinto any significant degree. As detailed herein, those MN-expressingnormal tissues include the human gastric mucosa and gallbladderepithelium, and some other normal tissues of the alimentary tract.Paradoxically, as shown herein, MN gene expression has been found to belost or reduced in carcinomas and other preneoplastic/neoplasticdiseases in some tissues that normally express MN, e.g., gastric mucosa.

In general, as elucidated by the examples herein, oncogenesis may besignified by the abnormal expression of MN protein. For example,oncogenesis may be signified: (1) when MN protein is present in a tissuewhich normally does not express MN protein to any significant degree;(2) when MN protein is absent from a tissue that normally expresses it;(3) when MN gene expression is at a significantly increased level, or ata significantly reduced level from that normally expressed in a tissue;or (4) when MN protein is expressed in an abnormal location within acell.

SUMMARY OF THE INVENTION

The discovery of the MN gene and protein and thus, of substantiallycomplementary MN genes and proteins encoded thereby, led to the findingthat the expression of MN proteins was associated with tumorigenicity.That finding resulted in the creation of methods that arediagnostic/prognostic for cancer and precancerous conditions. Methodsand compositions are provided for identifying the onset and presence ofneoplastic disease by detecting or detecting and quantitating abnormalMN gene expression in vertebrates.

Such abnormal MN gene expression can be detected or detected andquantitated by a variety of conventional assays in vertebrate samples,for example, by immunoassays using MN-specific antibodies to detect ordetect and quantitate MN antigen, by hybridization assays or by PCRassays, such as RT-PCR, using MN nucleic acids, such as, MN cDNA, todetect or detect and quantitate MN nucleic acids, such as, MN mRNA.

Vertebrate samples, preferably mammalian, more preferably human, caninclude tissue sections, tissue extracts, tissue smears, cells (whole orlysed), and cell extracts. MN antigen, preferably in a soluble form,more preferably as the MN extracellular domain, can also be found inbody fluids as an indicator of preneoplastic/neoplastic disease.

Preferred body fluids to assay according to this invention includeblood, serum, plasma, semen, breast exudate, saliva, tears, sputum,mucous, urine, gastric secretions, fecal suspensions, bile, lymph,cytosols, ascites, pleural effusions, amniotic fluid, bladder washes,bronchioalveolar lavages and cerebrospinal fluid.

Preferred samples and body fluids would in general depend upon the typeof preneoplastic/neoplastic disease for which the diagnosis/prognosis issought. For example, serum could be a more preferred body fluid to testfor renal cell carcinoma, breast, bladder or prostate cancer; however,urine could be a more preferred body fluid to test for urinary tracttumors, as renal cell carcinoma or bladder cancer. Still furtherpreferred for testing for abnormal MN gene expression associated withurinary tract cancer, especially bladder cancer, could be exfoliatedcells from urine. Mucous from the intestines and fecal suspensions couldbe preferred samples to test for duodenal, ileal, jejunal, and/orcolorectal cancers, whereas gastric secretions and bile could bepreferred body fluids to test respectively for stomach, andgallbladder/liver duct cancers.

The present invention is useful for detecting a wide variety ofneoplastic and/or preneoplastic diseases. Exemplary diseases includecarcinomas, such as mammary, lung, esophageal, prostate, bladder, renal,ovarian, gastrointestinal, uterine, uterine cervical, endometrial,squamous cell and adenosquamous carcinomas; and head and neck cancers;mesodermal tumors, such as neuroblastomas and retinoblastomas; sarcomas,such as osteosarcomas and Ewing's sarcoma; and melanomas. Of particularinterest are head and neck cancers, gynecologic cancers includingovarian, cervical, vaginal, endometrial and vulval cancers;gastrointestinal cancer, such as, esophageal, stomach, intestinal, colonand rectal cancers; urinary tract cancer, such as, bladder and kidneycancers; skin cancer; liver cancer; prostate cancer; lung cancer; andbreast cancer. Of still further particular interest are gynecologiccancers; breast cancer; urinary tract cancers, especially renal andbladder cancers; lung cancer; esphageal cancer; and colorectal cancer.Even further of particular interest are esophageal, lung, renal,colorectal, gynecologic and breast cancers. Gynecologic cancers ofparticular interest are carcinomas of the uterine cervix, endometriumand ovaries; more particularly such gynecologic cancers include cervicalsquamous cell carcinomas, adenosquamous carcinomas, adenocarcinomas aswell as gynecologic precancerous conditions, such as metaplasticcervical tissues and condylomas.

As indicated above, most normal tissues do not express MN protein. Thus,for most preneoplastic/neoplastic diseases, abnormal MN gene expressionis indicated by evidence of significant MN gene expression. The reversemay be true for preneoplastic/neoplastic disease of tissues thatnormally express MN protein, e.g., the gastric mucosa. MN protein isnormally expressed abundantly in the gastric mucosa and gallbladderepithelium. However, in the case of preneoplastic/neoplastic disease, MNprotein is absent from or at a significantly reduced level of expressionin the gastric mucosa. The absence or reduced MN expression in thegastric mucosa is thus indicative of oncogenesis, and signaled, forexample, by the absence or reduced amount of MN protein and/or MN genetranscription in vertebrate samples. Some evidence suggests in the caseof biliary epithelial tumors that MN expression becomes weaker withincrease of severity of dysplasia in premalignant lesions and decreaseof differentiation in carcinomas. [Saarnio et al., Gut, 41(3): 186(1997).] Also, the abnormal expression of MN may be signaled by itsexpression at an enhanced level at a different location than it isnormally expressed, e.g. other than in the basolateral surfaces of theepithelial cells of the ascending colon.

The immunoassays of this invention can be embodied in test kits whichcomprise MN proteins/polypeptides and/or MN-specific antibodies. Suchtest kits can be in solid phase formats, but are not limited thereto,and can also be in liquid phase format, and can be based onimmunohistochemical assays, ELISAS, particle assays, radiometric orfluorometric assays either unamplified or amplified, using, for example,avidin/biotin technology.

Test kits of this invention can comprise the nucleic acid probes of theinvention which are useful diagnostically/prognostically for neoplasticand/or preneoplastic disease. Preferred test kits comprise means fordetecting or measuring the hybridization of said probes to the MN geneor to the mRNA product of the MN gene, such as a visualizing means.

Further, this invention is directed to the MN gene, fragments thereofand the related cDNA which are useful, for example, as follows: 1) toproduce MN proteins/polypeptides by biochemical engineering; 2) toprepare nucleic acid probes to test for the presence of the MN gene incells of a subject; 3) to prepare appropriate polymerase chain reaction(PCR) primers for use, for example, in PCR-based assays or to producenucleic acid probes; 4) to identify MN proteins and polypeptides as wellas homologs or near homologs thereto; 5) to identify various mRNAstranscribed from MN genes in various tissues and cell lines, preferablyhuman; and 6) to identify mutations in MN genes. The invention furtherconcerns purified and isolated DNA molecules comprising the MN gene orfragments thereof, or the related cDNA or fragments thereof.

This invention in one aspect concerns isolated nucleic acid sequencesthat encode MN proteins or polypeptides wherein the nucleotide sequencesfor said nucleic acids are selected from the group consisting of:

(a) SEQ ID NO: 1;

(b) nucleotide (nt) sequences that hybridize specifically understringent conditions, for example, of 50% formamide at 42° C., to SEQ IDNO: 1 or to its complement;

(c) nucleotide sequences that differ from SEQ ID NO: 1 or from thenucleotide sequences of (b) in codon sequence because of the degeneracyof the genetic code. Further, such nucleic acid sequences are selectedfrom nucleotide sequences that but for the degeneracy of the geneticcode would hybridize to SEQ ID NO: 1 or to its complement understringent hybridization conditions.

This invention also concerns MN genomic sequences shown in FIG. 2A-F,that is, SEQ ID NO: 5, as well as nt sequences that hybridizespecifically to it or its complement under stringent conditions, whereinsuch nt sequences hybridize specifically to regions of the MN genomicsequence that are unique to MN, and do not hybridize specifically undersuch stringent conditions to nt sequences not unique to MN, such as, Alusequences or long terminal repeat (LTR) sequences, or would so hybridizeto SEQ ID NO: 5 or to its complement under such conditions, but for thedegeneracy of the genetic code. Degenerate variants of SEQ ID NOS: 1 and5 are within the scope of the invention.

Further, this invention concerns nucleic acid probes which are fragmentsof the isolated nucleic acids that encode MN proteins or polypeptidesand/or are from the MN genomic sequence which meet the abovehybridization criteria. Preferably said nucleic acid probes arecomprised of at least 25 nts, more preferably at least 27 nts, stillmore preferably at least 29 nts, further preferably at least 50 nts,further more preferably at least 100 nts, and even more preferably atleast 150 nts.

Still further, this invention is directed to isolated nucleic acidscontaining at least twenty-five nucleotides selected from the groupconsisting of:

(a) SEQ ID NOS: 1, 3, 5, 7, 8, 17, 18, 27-49, 55-66, 88-96, 110 and 115and sequences that are complementary to SEQ ID NOS: 1, 3, 5, 7, 8, 17,18, 27-49, 55-66, 88-96, 110 and 115;

(b) nucleotide sequences that hybridize under standard stringenthybridization conditions, for example, of 50% formamide at 42° C., toone or more of the following nucleotide sequences: SEQ ID NOS: 1, 3, 5,7, 8, 17, 18, 27-49, 55-66, 88-96, 110 and 115 and the respectivecomplements of SEQ ID NOS: 1, 3, 5, 7, 8, 17, 18, 27-49, 55-66, 88-96,110 and 115, but do not hybridize specifically under such stringentconditions to nt sequences not unique to MN, such as, Alu sequences orLTR sequences; and

(c) nucleotide sequences that differ from the nucleotide sequences of(a) and (b) in codon sequence because of the degeneracy of the geneticcode. The invention also concerns nucleic acids that but for thedegeneracy of the genetic code would hybridize specifically to thenucleic acids of (a) and (b) under standard stringent hybridizationconditions. Further this invention concerns nucleic acids of (b) and (c)that hybridize partially or wholly to the non-coding regions of SEQ IDNO: 5 or its complement as, for example, sequences that function asnucleic acid probes to identify MN nucleic acid sequences, but do nothybridize specifically to regions of the MN genomic region that are notunique to MN. Conventional technology can be used to determine whetherthe nucleic acids of (b) and (c) or of fragments of SEQ ID NO: 5 areuseful to identify MN nucleic acid sequences, for example, as outlinedin Benton and Davis, Science, 196: 180 (1977) and Fuscoe et al.Genomics. 5: 100 (1989). In general, such nucleic acids are preferablyat least 25 nts, more preferably at least 27 nts, still more preferablyat least 29 nts, further preferably at least 50 nts, and still morepreferably at least 100 nts. An exemplary and preferred nucleic acidprobe is SEQ ID NO: 55 (a 470 bp probe useful in RNase portectionassays).

Fragments of the isolated nucleic acids of the invention, can also beused as PCR primers, e.g. in RT-PCR, to detect MN expression, to amplifysegments of MN genes, and to identify mutations in MN genes. Typically,said PCR primers are olignucleotides, preferably having a length ofabout 14 nts to about 25 nts, more preferably from about 16 to 20 nts,but they may be considerably longer. Exemplary primers may be from about16 nucleotides to about 50 nucleotides, preferably from about 17nucleotides to about 45 nucleotides.

Further, the invention concerns the use of such PCR primers in methodsto detect mutations in an isolated MN gene and/or fragment(s) thereof.For example, such methods can comprise amplifying one or morefragment(s) of an MN gene by PCR, and determining whether any of saidone or more fragments contain mutations, by, for example, comparing thesize of the amplified fragments to those of similarly amplifiedcorresponding fragments of MN genes known to be normal, by using aPCR-single-strand conformation polymorphism assay or a denaturinggradient gel electrophoretic assay.

This invention also concerns nucleic acids which encode MN proteins orpolypeptides that are specifically bound by monoclonal antibodiesdesignated M75 that are produced by the hybridoma VU-M75 deposited atthe American Type Culture Collection (ATCC), now at 10801 UniversityAvenue, Manassas, Va. 20110-22209 (USA) under ATCC No. HB 11128, and/orby monoclonal antibodies designated MN12 produced by the hybridoma MN12.2.2 deposited at the ATCC under ATCC No. HB 11647.

This invention further concerns isolated nucleic acids containing atleast sixteen nts, preferably at least twenty-seven nts, more preferablyat least twenty-nine nts, still more preferably at least fifty nts,wherein said nucleic acid is selected from the group consisting of:

(a) the MN nucleic acids contained in plasmids A4a, XE1 and XE3 whichwere deposited at the ATCC under the respective ATCC Nos. 97199, 97200,and 97198;

(b) nucleic acids that hybridize specifically under stringent conditionsto regions of those MN nucleic acids of (a) that are unique to the MNgene, that do not hybridize specifically under such stringent conditionsto nt sequences that are not unique to MN, such as, Alu sequences or LTRsequences; and

(c) nucleic acids that differ from the nucleic acids of (a) or (b) incodon sequence due to the degeneracy of the genetic code. Such isolatednucleic acids, for example, can be polymerase chain reaction (PCR)primers.

The invention further concerns isolated nucleic acids that code for anMN protein, MN fusion protein or MN polypeptide that is operativelylinked to an expression control sequence within a vector; unicellularhosts, prokaryotic or eukaryotic, that are transformed or transfectedtherewith; and methods of recombinantly producing MN proteins, MN fusionproteins and MN polypeptides comprising transforming or transfectingunicellular hosts with said nucleic acid operatively linked to anexpression control sequence, culturing said transformed or transfectedunicellular hosts so that said MN proteins, fusion proteins orpolypeptides are expressed, and extracting and isolating said MN proteinfusion protein or polypeptide.

Recombinant nucleic acids that encode MN fusion proteins are claimed ascomprising an MN protein or MN polypeptide and a non-MN protein orpolypeptide wherein the nucleotide sequence for the portion of thenucleic acid encoding the MN protein or polypeptide is selected from thegroup consisting of:

(a) SEQ ID NO: 1;

(b) nucleotide sequences that hybridize under stringent conditions toSEQ ID NO: 1 or to its complement; and

(c) degenerate variants of SEQ ID NO: 1, and of the nucleotide sequencesof (b); wherein the nucleic acid encoding said MN protein or polypeptidepreferably contains at least twenty-five nts.

Said non-MN protein or polypeptide may preferably be nonimmunogenic tohumans and not typically reactive to antibodies in human body fluids.Examples of such a DNA sequence is the alpha-peptide coding region ofbeta-galactosidase and a sequence coding for glutathione S-transferase(GST) or a fragment thereof. However, in some instances, a non-MNprotein or polypeptide that is serologically active, immunogenic and/orantigenic may be preferred as a fusion partner to a MN antigen. Further,claimed herein are such recombinant fusion proteins/polypeptides whichare substantially pure and non-naturally occurring. Exemplary fusionproteins of this invention are GST-MN, MN-Fc and MN-PA, described infra.

In HeLa and in tumorigenic HeLa×fibroblast hybrid (H/F-T) cells, MNprotein is manifested as a “twin” protein p54/58N; it is glycosylatedand forms disulfide-linked oligomers. As determined by electrophoresisupon reducing gels, MN proteins have molecular weights in the range offrom about 40 kd to about 70 kd, preferably from about 45 kd to about 65kd, more preferably from about 48 kd to about 58 kd. Upon non-reducinggels, MN proteins in the form of oligomers have molecular weights in therange of from about 145 kd to about 220 kd, preferably from about 150 toabout 200 kd, still more preferably from about 150 to about 155 kd, andfurther preferably from about 152 to about 154 kd. A predicted aminoacid sequence for a preferred MN protein of this invention is shown inFIG. 1 [SEQ. ID. NO. 2].

Other particular MN proteins or polypeptides are exemplified by theputative MN signal peptide shown as the first thirty-seven amino acidsin FIG. 1 [SEQ ID NO: 6], preferred MN antigen epitopes [represented by,e.g., SEQ ID NOS: 10-16 and 98-103], and domains of the MN proteinrepresented in FIG. 1 amino acids (aa) 38-414 (SEQ ID NO: 87; theextracellular domain), aa 415-434 (SEQ ID NO: 52; the transmembranedomain), aa 435-459 (SEQ ID NO: 53; the intracellular domain), aa 53-111(SEQ ID NO: 50; the proteoglycan-like domain); and aa 135-391 (SEQ IDNO: 51; the CA domain). Also, of particular importance is the regionwithin the proteoglycan-like domain, aa 61-96 (SEQ ID NO: 97) whichcontains a 6-fold tandem repeat of 6 amino acids, and within which theepitope for the M75 MAb resides in at least two copies, and within whichthe MN binding site is considered to be located. An alternative MNbinding site may be located in the CA domain.

Identified herein is the location of the MN protein binding site. Alsoidentified are MN oligopeptides that compete for attachment to cellswith immobilized MN protein. Such oligopeptides prevent cell-celladhesion and the formation of intercellular contacts.

Disclosed herein are cell adhesion assay methods that are used toidentify binding site(s) on the MN protein to which vertebrate cells,preferably mammalian cells bind. Such a MN binding site is thenidentified as a therapeutic target which can be blocked withproteins/polypeptides, MN-specific antibodies or organic molecules thatspecifically bind to said site.

Further disclosed are therapeutic methods to treat patients withpreneoplastic/neoplastic disease associated with or characterized byabnormal MN expression, which methods are based on blocking said MNbinding site with proteins/polypeptides, MN-specific antibodies ororganic molecules, that bind specifically to said binding site. Thegrowth of a vertebrate preneoplastic/neoplastic cell that abnormallyexpresses MN protein can be inhibited by administering suchproteins/polypeptides, MN-specific antibodies or organic molecules in atherapeutically effective amount in a physiologically acceptableformulation. Such a preferred therapeutic protein/polypeptide is hereinconsidered to comprise an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 107-109. Such heptapeptides are considered tobe comprised by MN protein partner(s). Blocking the interaction betweenMN protein and its binding partner(s), is expected to lead to a decreaseof tumor growth.

A preferred MN-specific antibody for such a therapeutic use would be aMN-specific antibody that has an epitope selected from the groupconsisting of SEQ ID NOS: 10 and 98-103. The M75 MAb is an especiallypreferred MN-specific antibody for use in cancer therapy. Suchtherapeutic methods are preferably directed to mammals and morepreferably to humans.

Also provided are therapeutic methods wherein the growth of avertebrate, preferably mammalian, more preferably human, preneoplasticor neoplastic cell that abnormally expresses MN protein is inhibited.Said methods comprise transfecting said cell with a vector comprising anexpression control sequence operatively linked to a nucleic acidencoding the variable domains of an MN-specific antibody, wherein saiddomains are separated by a flexible linker peptide, preferably SEQ IDNO: 116. Preferably said expression control sequence comprises the MNgene promoter.

Alternatively, such therapeutic methods comprise transfecting said cellwith a vector comprising a nucleic acid that encodes a cytotoxicprotein/polypeptide, such as HSVtk, operatively linked to the MN genepromoter. Such a therapeutic vector may also comprise a nucleic acidencoding a cytokine, such as, IL-2 or IFN.

Still further, such therapeutic/prophylactic methods comprise inducingMN-specific antibody production within a patient by injecting saidpatient with an anti-idiotype antibody to a MN-specific antibody. Stillfurther, such therapeutic methods can include treating a patient with apreneoplastic and/or neoplastic disease characterized by abnormal MNexpression by administering to said patient a therapeutically effectiveamount of an anti-anti-idiotype MN-specific antibody serum, alone or incombination with one or more cytokines, preferably with IFN and/or IL-2.

The invention further relates to the biochemical engineering of the MNgene, fragments thereof or related cDNA. For example, said gene or afragment thereof or related cDNA can be inserted into a suitableexpression vector, wherein it is operatively linked to an expressioncontrol sequence; host cells, preferably unicellular, can be transformedor transfected with such an expression vector; and an MNprotein/polypeptide, preferably an MN protein, is expressed therein.Such a recombinant protein or polypeptide can be glycosylated ornonglycosylated, preferably glycosylated, and can be purified tosubstantial purity. The invention further concerns MNproteins/polypeptides which are synthetically or otherwise biologicallyprepared.

Disclosed herein are biologically active MN proteins and MN polypeptidesthat are useful as vaccines to protect vertebrates, preferably mammals,more preferably humans, against neoplastic diseases associated withabnormal MN expression. Such vaccines are also useful to boost apatient's immunity to such a disease. Such vaccines can alternativelycomprise an anti-idiotype MN-specific antibody. Such vaccines areadministered in a therapeutically effective amount in a physiologicallyacceptable formulation.

A preferred biologically active MN protein or MN polypeptide isconsidered to comprise an amino acid sequence from SEQ ID NOS: 50, 51,or 97. Such a preferred MN protein or MN polypeptide may comprise orhave an amino acid sequence selected from the group consisting of SEQ IDNOS: 10 and 97-106. MN-specific antibodies that bind to suchbiologically active MN proteins and polypeptides are also preferred.Particularly preferred are such MN proteins and MN polypeptides that arespecifically bound by the M75 MAb, or the MN12 MAb, preferably by theM75 MAb, such as, the synthetic peptides represented by SEQ ID NOS:104-106.

Also disclosed are anti-idiotype antibodies to MN-specific antibodies,and anti-anti-idiotype antibodies thereto, polyclonal or monoclonal.Such anti-idiotype antibodies are useful as vaccines, and theanti-anti-idiotype antibody sera are therapeutically useful againstneoplastic diseases associated with abnormal MN expression.

MN proteins/polypeptides can be used in assays to detect MN antigen inpatient samples and in serological assays to test for MN-specificantibodies. MN proteins/polypeptides of this invention are serologicallyactive, immunogenic and/or antigenic. They can further be used asimmunogens to produce MN-specific antibodies, polyclonal and/ormonoclonal, as well as an immune T-cell response.

The invention is directed to MN-specific antibodies, which can be useddiagnostically/prognostically and may be used therapeutically. Preferredaccording to this invention are MN-specific antibodies reactive with theepitopes represented respectively by the amino acid sequences of the MNprotein shown in FIG. 1 as follows: aa 62-67 (SEQ ID NO: 10); aa 61-66,aa 79-84, aa 85-90 and aa 91-96 (SEQ ID NO: 98); aa 62-65, aa 80-83, aa86-89 and aa 92-95 (SEQ ID NO: 99); aa 62-66, aa 80-84, aa 86-90 and aa92-96 (SEQ ID NO: 100); aa 63-68 (SEQ ID NO: 101); aa 62-68 (SEQ ID NO:102); aa 82-87 and aa 88-93 (SEQ ID NO: 103); aa 55-60 (SEQ ID NO: 11);aa 127-147 (SEQ ID NO: 12); aa 36-51 (SEQ ID NO: 13); aa 68-91 (SEQ IDNO: 14); aa 279-291 (SEQ ID NO: 15); and aa 435450 (SEQ ID NO: 16). Morepreferred are antibodies reactive with epitopes represented by SEQ IDNOS: 10, 98-103, 11 and 12. Still more preferred are antibodies reactivewith the epitopes represented by SEQ. ID NOS: 10, 98-103 and 11. Mostpreferred are monoclonal antibodies reactive with the epitoperepresented by SEQ ID NOS: 10 and 98-103.

Also preferred according to this invention are antibodies preparedagainst recombinantly produced MN proteins as, for example, GST-MN, MN20-19, MN-Fc and MN-PA. Also preferred are MN-specific antibodiesprepared against glycosylated MN proteins, such as, MN 20-19 expressedin baculovirus infected Sf9 cells.

A hybridoma that produces a representative MN-specific antibody, themonoclonal antibody M75 (Mab M75), was deposited at the ATCC underNumber HB 11128 as indicated above. The M75 antibody was used todiscover and identify the MN protein and can be used to identify readilyMN antigen in Western blots, in radioimmunoassays andimmunohistochemically, for example, in tissue samples that are fresh,frozen, or formalin-, alcohol-, acetone- or otherwise fixed and/orparaffin-embedded and deparaffinized. Another representative MN-specificantibody, Mab MN12, is secreted by the hybridoma MN 12.2.2, which wasdeposited at the ATCC under the designation HB 11647.

MN-specific antibodies can be used, for example, in laboratorydiagnostics, using immunofluorescence microscopy or immunohistochemicalstaining; as a component in immunoassays for detecting and/orquantitating MN antigen in, for example, clinical samples; as probes forimmunoblotting to detect MN antigen; in immunoelectron microscopy withcolloid gold beads for localization of MN proteins and/or polypeptidesin cells; and in genetic engineering for cloning the MN gene orfragments thereof, or related cDNA. Such MN-specific antibodies can beused as components of diagnostic/prognostic kits, for example, for invitro use on histological sections; such antibodies can also and usedfor in vivo diagnostics/prognostics, for example, such antibodies can belabeled appropriately, as with a suitable radioactive isotope, and usedin vivo to locate metastases by scintigraphy. Further such antibodiesmay be used in vivo therapeutically to treat cancer patients with orwithout toxic and/or cytostatic agents attached thereto. SuchMN-specific antibodies for therapeutic use may be directed to theextracellular (EC), transmembrane (TM) and/or intracellular (IC) domainsof MN. Further, such antibodies can be used in vivo to detect thepresence of neoplastic and/or preneoplastic disease. Still further, suchantibodies can be used to affinity purify MN proteins and polypeptides.

This invention also concerns methods of treating neoplastic diseaseand/or preneoplastic disease comprising inhibiting the expression of MNgenes by administering antisense nucleic acid sequences that arecomplementary to mRNA transcribed from MN genes. Said antisense nucleicacid sequences are those that hybridize specifically to such mRNA understringent hybridization conditions. Preferred are antisense nucleic acidsequences that are complementary to sequences at the 5′ end of the MNcDNA sequence shown in FIG. 1, more preferably to the 5′ leader sequenceof said mRNA. Preferably said antisense nucleic acid sequences areoligonucleotides.

This invention also concerns vaccines comprising an immunogenic amountof one or more substantially pure MN proteins and/or polypeptides oranti-idiotype antibodies (including variations thereof), dispersed in aphysiologically acceptable, nontoxic vehicle, which amount is effectiveto immunize a vertebrate, preferably a mammal, more preferably a human,against a preneoplastic/neoplastic disease associated with theexpression of MN proteins. Said proteins can be recombinantly,synthetically or otherwise biologically produced. A particular use ofsaid vaccine would be to prevent recidivism and/or metastasis. Forexample, it could be administered to a patient who has had anMN-carrying tumor surgically removed, to prevent recurrence of thetumor.

The MN gene promoter is characterized herein. The identification of thebinding site for a repressor of MN transcription is disclosed.Identification of the protein that binds to the repressor andmodification of its binding properties is another route to modulate MNexpression leading to cancer therapies. Suppression of MN expression intumor cells by over expression of a negative regulator is expected tolead to a decrease of tumor growth.

Abbreviations

The following abbreviations are used herein:

-   aa—amino acid-   ATCC—American Type Culture Collection-   bp—base pairs-   BLV—bovine leukemia virus-   BSA—bovine serum albumin-   BRL—Bethesda Research Laboratories-   CA—carbonic anhydrase-   CAM—cell adhesion molecule-   CARP—carbonic anhydrase related protein-   CAT—chloramphenicol acetyltransferase-   Ci—curie-   cm—centimeter-   CMV—cytomegalovirus-   cpm—counts per minute-   C-terminus—carboxyl-terminus-   CTL—cytotoxic T lymphocytes-   ° C.—degrees centigrade-   DEAE—diethylaminoethyl-   DMEM—Dulbecco modified Eagle medium-   ds—double-stranded-   EDTA—ethylenediaminetetraacetate-   EGF—epidermal growth factor-   EIA—enzyme immunoassay-   ELISA—enzyme-linked immunosorbent assay-   EMSA—electrophoretic mobility shift assay-   F—fibroblasts-   FACS—cytofluorometric study-   FCS—fetal calf serum-   FITC—fluorescein isothiocyanate-   FTP—DNase 1 footprinting analysis-   GST-MN—fusion protein MN glutathione S-transferase-   GVC—ganciclovir-   H—HeLa cells-   H-E—haematoxylin-eosin-   HEF—human embryo fibroblasts-   HeLa K—standard type of HeLa cells-   HeLa S—Stanbridge's mutant HeLa D98/AH.2-   H/F-T—hybrid HeLa fibroblast cells that are tumorigenic; derived    from HeLa D98/AH.2-   H/F-N—hybrid HeLa fibroblast cells that are nontumorigenic; derived    from HeLa D98/AH.2-   HPV—Human papilloma virus-   HRP—horseradish peroxidase-   HSV—Herpes simplex virus-   IC—intracellular-   IFN—interferon-   IL-2-interleukin-2-   Inr—initiator-   IPTG—isopropyl-beta-D-thiogalacto-pyranoside-   kb—kilobase-   kbp—kilobase pairs-   kd or kDa—kilodaltons-   KS—keratan sulphate-   LCMV—lymphocytic choriomeningitis virus-   LTR—long terminal repeat-   M—molar-   mA—milliampere-   MAb—monoclonal antibody-   MCSF—macrophage colony stimulating factor-   ME—mercaptoethanol-   MEM—minimal essential medium-   min.—minute(s)-   mg—milligram-   ml—milliliter-   mM—millimolar-   MMC—mitomycin C-   mmol—millimole-   MLV—murine leukemia virus-   N—normal concentration-   NEG—negative-   ng—nanogram-   nm—nanometer-   nt—nucleotide-   N-terminus—amino-terminus-   ODN—oligodeoxynucleotide-   ORF—open reading frame-   PA—Protein A-   PBS—phosphate buffered saline-   PCR—polymerase chain reaction-   PEST—combination of one-letter abbreviations for proline, glutamic    acid, serine, threonine-   PG—proteoglycan-   pl—isoelectric point-   PMA—phorbol 12-myristate 13-acetate-   POS—positive-   Py—pyrimidine-   RACE—rapid amplification of cDNA ends-   RCC—renal cell carcinoma-   RIA—radioimmunoassay-   RIP—radioimmunoprecipitation-   RIPA—radioimmunoprecipitation assay-   RNP—RNase protection assay-   RT-PCT—reverse transcription polymerase chain reaction-   SAC—Staphylococcus aureus cells-   S. aureus—Staphylococcus aureus-   sc—subcutaneous-   SDRE—serum dose response element-   SDS—sodium dodecyl sulfate-   SDS-PAGE—sodium dodecyl sulfate-polyacrylamide gel electrophoresis-   SINE—short interspersed repeated sequence-   SP—signal peptide-   SP-RIA—solid-phase radioimmunoassay-   SSDS—synthetic splice donor site-   SSH—subtractive suppressive PCR-   SSPE—NaCl (0.18 M), sodium phosphate (0.01 M), EDTA (0.001 M)-   TBE—Tris-borate/EDTA electrophoresis buffer-   TC—tissue culture-   TCA—trichloroacetic acid-   TC media—tissue culture media-   TC—tissue culture-   tk—thymidine kinase-   TM—transmembrane-   TMB—tetramethylbenzidine-   Tris—tris(hydroxymethyl)aminomethane-   μCi—microcurie-   μg—microgram-   μl—microliter-   μM—micromolar-   VSV—vesicular stomatitis virus-   VV—vaccinia virus

X-MLV—xenotropic murine leukemia virus Cell Lines AGS cell line derivedfrom a primary adenogastric carcinoma [Barranco and Townsend, CancerRes., 43: 1703 (1983) and Invest. New Drugs, 1: 117 (1983)]; availablefrom the ATCC under CRL-1739; BL-3 bovine B lymphocytes [ATCC CRL-8037;leukemia cell suspension; J. Natl. Cancer Inst. (Bethesda) 40: 737(1968)]; C33 a cell line derived from a human cervical carcinoma biopsy[Auersperg, N., J. Nat'l. Cancer Inst. (Bethesda), 32: 135-148 (1964)];available from the ATCC under HTB-31; C33A human cervical carcinomacells [ATCC HTB-31; J. Natl. Cancer Inst. (Bethesda) 32: 135 (1964)];COS simian cell line [Gluzman, Y., Cell, 23: 175 (1981)]; HeLa Kstandard type of HeLa cells; aneuploid, epithelial-like cell lineisolated from a human cervical adenocarcinoma [Gey et al., Cancer Res.,12: 264 (1952); Jones et al., Obstet. Gynecol., 38: 945-949 (1971)]obtained from Professor B. Korych, [Institute of Medical Microbiologyand Immunology, Charles University; Prague, Czech Republic]; HeLa MutantHeLa clone that is hypoxanthine D98/AH.2 guanine phosphoribosyltransferase-deficient (HGPRT⁻) (also kindly provided by Eric J.Stanbridge [Department of HeLa s) Microbiology, College of Medicine,University of California, Irvine, CA (USA)] and reported in Stanbridgeet al., Science 215: 252-259 (15 Jan. 1982); parent of hybrid cellsH/F-N and H/F-T, also obtained from E. J. Stanbridge; KATO III cell lineprepared from a metastatic form of a gastric carcinoma [Sekiguichi etal., Japan J. Exp. Med., 48: 61 (1978)]; available from the ATCC underHTB-103; NIH-3T3 murine fibroblast cell line reported in Aaronson,Science, 237: 178 (1987); QT35 quail fibrosarcoma cells [ECACC:93120832; Cell, 11: 95 (1977)]; Raj human Burkitt's lymphoma cell line[ATCC CCL-86; Lancet, 1: 238 (1964)]; Rat2TK⁻ cell line (rat embryo,thymidine kinase mutant) was derived from a subclone of a5′-bromo-deoxyuridine resistant strain of the Fischer rat fibroblast3T3-like cell line Rat1; the cells lack appreciable levels of nuclearthymidine kinase [Ahrens, B., Virology, 113: 408 (1981)]; SiHa humancervical squamous carcinoma cell line [ATCC HTB- 35; Friedl et al.,Proc. Soc. Exp. Biol. Med., 135: 543 (1990)]; XC cells derived from arat rhabdomyosarcoma induced with Rous sarcoma virus-induced rat sarcoma[Svoboda, J., Natl. Cancer Center Institute Monograph No. 17, IN:“International Conference on Avian Tumor Viruses” (J. W. Beard ed.), pp.277-298 (1964)], kindly provided by Jan Svoboda [Institute of MolecularGenetics, Czechoslovak Academy of Sciences; Prague, Czech Republic]; andCGL1 H/F-N hybrid cells (HeLa D98/AH.2 derivative); CGL2 H/F-N hybridcells (HeLa D98/AH.2 derivative); CGL3 H/F-T hybrid cells (HeLa D98/AH.2derivative); CGL4 H/F-T hybrid cells (HeLa D98/Ah.2 derivative).

Nucleotide and Amino Acid Sequence Symbols

The following symbols are used to represent nucleotides herein: BaseSymbol Meaning A adenine C cytosine G guanine T thymine U uracil Iinosine M A or C R A or G W A or T/U S C or G Y C or T/U K G or T/U V Aor C or G H A or C or T/U D A or G or T/U B C or G or T/U N/X A or C orG or T/U

There are twenty main amino acids, each of which is specified by adifferent arrangement of three adjacent nucleotides (triplet code orcodon), and which are linked together in a specific order to form acharacteristic protein. A three-letter or one-letter convention is usedherein to identify said amino acids, as, for example, in FIG. 1 asfollows: 3 Ltr. 1 Ltr. Amino acid name Abbrev. Abbrev. Alanine Ala AArginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys CGlutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Unknown or other X

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C provides the nucleotide sequence for a MN cDNA [SEQ ID NO: 1]clone isolated as described herein. FIG. 1 A-C also sets forth thepredicted amino acid sequence [SEQ ID NO: 2] encoded by the cDNA.

FIG. 2A-F provides a 10,898 bp complete genomic sequence of MN [SEQ IDNO: 5]. The base count is as follows: 2654 A; 2739 C; 2645 G; and 2859T. The 11 exons are in general shown in capital letters, but exon 1 isconsidered to begin at position 3507 as determined by RNase protectionassay.

FIG. 3 is a restriction map of the full-length MN cDNA. The open readingframe is shown as an open box. The thick lines below the restriction mapillustrate the sizes and positions of two overlapping cDNA clones. Thehorizontal arrows indicate the positions of primers R1 [SEQ ID NO: 7]and R2 [SEQ ID NO: 8] used for the 5′ end RACE. Relevant restrictionsites are BamHI (B), EcoRV (V), EcoRI (E), PstI (Ps), PvuII (Pv).

FIG. 4 schematically represents the 5′ MN genomic region of a MN genomicclone wherein the numbering corresponds to transcription initiationsites estimated by RACE.

FIG. 5 provides an exon-intron map of the human MN/CA IX gene. Thepositions and sizes of the exons (numbered, filled boxes), Alu repeatelements (open boxes) and an LTR-related sequence (first unnumbered greyor stippled box) are adjusted to the indicated scale. The exonscorresponding to individual MN/CA IX protein domains are enclosed indashed frames designated PG (proteoglycan-like domain), CA (carbonicanhydrase domain), TM (transmembrane anchor) and IC (intracytoplasmictail). Below the map, the alignment of amino acid sequences illustratesthe extent of homology between the MN/CA IX protein PG region (aa53-111) [SEQ ID NO: 50] and the human aggrecan (aa 781-839) [SEQ ID NO:54].

FIG. 6 is a nucleotide sequence for the proposed promoter of the humanMN gene [SEQ ID NO: 27]. The nucleotides are numbered from thetranscription initiation site according to RNase protection assay.Potential regulatory elements are overlined. Transcription start sitesare indicated by asterisks (RNase protection) and dots (RACE) above thecorresponding nucleotides. The sequence of the 1st exon begins under theasterisks. FTP analysis of the MN4 promoter fragment revealed 5 regions(I-V) protected at both the coding and noncoding strands, and tworegions (VI and VII) protected at the coding strand but not at thenoncoding strand.

FIG. 7 provides a schematic of the alignment of MN genomic clonesaccording to their position related to the transcription initiationsite. All the genomic fragments except Bd3 were isolated from a lambdaFIX II genomic library derived from HeLa cells. Clone Bd3 was derivedfrom a human fetal brain library.

FIG. 8 schematically represents the MN protein structure. Theabbreviations are the same as used in FIG. 5. The scale indicates thenumber of amino acids.

FIG. 9 outlines an experiment designed to explain the mechanism ofreversion of NIH3T3 cells transformed with the MN coding sequence(pGD.MN).

DETAILED DESCRIPTION

The terms “MN/CA IX” and “MN/CA9” are herein considered to be synonymsfor MN. Also, the G250 antigen is considered to refer to MNprotein/polypeptide. [Uemura et al., J. Urol. 154 (4 Suppl.): 377(Abstract 1475; 1997).]

The MN gene is shown herein to be organized into 11 exons and 10introns. Described herein is the cloning and sequencing of the MN cDNAand genomic sequences, and the genetic engineering of MN proteins—suchas the GST-MN, MN-PA, MN-Fc and MN 20-19 proteins. The recombinant MNproteins can be conveniently purified by affinity chromatography.

MN/CA IX was first identified in HeLa cells, derived from humancarcinoma of cervix uteri, as both a plasma membrane and nuclear proteinwith an apparent molecular weight of 58 and 54 kilodaltons (kDa) asestimated by Western blotting. It is N-glycosylated with a single 3 kDacarbohydrate chain and under non-reducing conditions forms S-S-linkedoligomers [Pastorekova et al., Virology. 187: 620-626 (1992); Pastoreket al., Oncogene. 9: 2788-2888 (1994)]. MN/CA IX is a transmembraneprotein located at the cell surface, although in some cases it has beendetected in the nucleus [Zavada et al., Int. J. Cancer. 54: 268-274(1993); Pastorekova et al., supra].

MN is manifested in HeLa cells by a twin protein, p54/58N. Immunoblotsusing a monoclonal antibody reactive with p54/58N (MAb M75) revealed twobands at 54 kDa and 58 kDa. Those two bands may correspond to one typeof protein that most probably differs by post-translational processing.Herein, the phrase “twin protein” indicates p54/58N.

MN expression is considered to be diagnostic/prognostic forpreneoplastic/neoplastic disease. The MN twin protein, p54/58N, wasfound to be expressed in HeLa cells and in Stanbridge's tumorigenic(H/F-T) hybrid cells [Stanbridge et al., Somatic Cell Genet., 7: 699-712(1981); and Stanbridge et al., Science, 215: 252-259 (1982)] but not infibroblasts or in non-tumorigenic (H/F—N) hybrid cells [Stanbridge etal., id.]. In early studies reported in Zavada et al. WO 93/18152,supra, MN proteins were found in immunoblots prepared from humanovarian, endometrial and uterine cervical carcinomas, and in some benignneoplasias (as mammary papilloma) but not from normal ovarian,endometrial, uterine or placental tissues.

Immunohistochemical studies with the M75 MAb of cervical carcinomas anda PCR-based (RT-PCR) survey of renal cell carcinomas have identified MNexpression as closely associated with those cancers and indicates thatMN has utility as a tumor biomarker. [Liao et al., Am. J. Pathol., 145:598-609 (1994); Liao et al., Cancer Epidemiol. Biomarkers Prev., 5:549-557 (1996); McKiernan et al., Cancer Res., 57: 2362-2365 (1997).]

Many studies have confirmed the diagnostic/prognostic utility of MN. Thefollowing articles discuss the use of the MN-specific MAb M75 indiagnosing/prognosing precancerous and cancerous cervical lesions: Leff,D. N., “Half a Century of HeLa Cells: Transatlantic Antigen EnhancesReliability of Cervical Cancer Pap Test, Clinical Trials Pending,”BioWorld® Today: The Daily Biotechnology Newspaper, 9(55) (Mar. 24,1998); Stanbridge, E. J., “Cervical marker can help resolve ambigous Papsmears,” Diagnostics Intelligence, 10(5): 11 (1998); Liao andStanbridge, “Expression of the MN Antigen in Cervical PapanicolaouSmears Is an Early Diagnostic Biomarker of Cervical Dysplasia,” CancerEpidemiology, Biomarkers & Prevention, 5: 549-557 (1996); Brewer et al.,“A Study of Biomarkers in Cervical Carcinoma and Clinical Correlation ofthe Novel Biomarker MN,” Gynecologic Oncology, 63: 337-344 (1996); andLiao et al., “Identification of the MN Antigen as a Diagnostic Biomarkerof Cervical Intraepithelial Squamous and Glandular Neoplasia andCervical Carcinomas,” American Journal of Pathology, 145(3): 598-609(1994).

Premalignant and Malignant Colorectal Lesions. MN has been detected innormal gastric, intestinal, and biliary mucosa. [Pastorekova et al.,Gastroenterology, 112: 398-408 (1997).] Immunohistochemical analysis ofthe normal large intestine revealed moderate staining in the proximalcolon, with the reaction becoming weaker distally. The staining wasconfined to the basolateral surfaces of the cryptal epithelial cells,the area of greatest proliferative capacity. As MN is much more abundantin the proliferating cryptal epithelium than in the upper part of themucosa, it may play a role in control of the proliferation anddifferentiation of intestinal epithelial cells. Cell proliferationincreases abnormally in premalignant and malignant lesions of thecolorectal epithelium, and therefore, is considered an indicator ofcolorectal tumor progression. [Risio, M., J. Cell Biochem, 16G: 79-87(1992); and Moss et al., Gastroenterology, 111: 1425-1432 (1996).]

Renal Cell Carcinoma (RCC. Nakagawa et al., J. Urol., 159(5)(Suppl.):Abstract 720 (May 1998), investigated MN expression in renal cellcarcinoma (RCC) by immunostaining using the MN-specific MAb G250 andRT-PCR using primers derived from the MN cDNA. “Immunohistochemistrywith MAbG250 resulted in strong and homogeneous expression in 131/145(90.3%) of RCC, whereas no expression was observed in correspondingnormal tissues. RT-PCR analyses of RCC frozen specimens resulted in theclear detection of MN mRNA signals, however . . . results were notidentical to immunohistochemistry . . . . PCR analysis of peripheralblood samples from the patients with progressive disease revealed thepresence of circulating cancer cells in the blood.”

Anticancer Drugs and Antibodies that Block Interaction of MN Protein andReceptor Molecules

MN protein is considered to be a uniquely suitable target for cancertherapy for a number of reasons including the following. (1) It islocalized on the cell surface, rendering it accessible. (2) It isexpressed in a high percentage of human carcinomas (e.g., uterinecervical, renal, colon, breast, esophageal, lung, head and neckcarcinomas, among others), but is not normally expressed to anysignificant extent in the normal tissues from which such carcinomasoriginate.

(3) It is normally expressed only in the stomach mucosa and in someepithelia of the digestive tract (epithelium of gallbladder and smallintestine). An anatomic barrier thereby exists between the MN-expressingpreneoplastic/neoplastic and MN-expressing normal tissues. Drugs,including antibodies, can thus be administered which can reach tumorswithout interfering with MN-expressing normal tissues.

(4) MAb M75 has a high affinity and specificity to MN protein. (5) MNcDNA and MN genomic clones which encompass the protein-coding and generegulatory sequences have been isolated. (6) MN-specific antibodies havebeen shown to have among the highest tumor uptakes reported in clinicalstudies with antitumor antibodies in solid tumors, as shown for theMN-specific chimeric antibody G250 in animal studies and in phase Iclinical trials with renal carcinoma patients. [Steffens et al., J.Clin. Oncol., 15: 1529 (1997).] Also, MN-specific antibodies have lowuptake in normal tissues.

Data, e.g. as presented herein, are consistent with the following theoryconcerning how MN protein acts in normal tissues and inpreneoplastic/neoplastic tissues. In normal tissues (e.g., in stomachmucosa), MN protein is considered to be a differentiation factor. Itbinds with its normal receptor S (for stomach). Stomach carcinomas havebeen shown not to contain MN protein.

Ectopic expression of MN protein in other tissues causes malignantconversion of cells. Such ectopic expression is considered to be causedby the binding of MN protein with an alternative receptor H (for HeLacells), coupled to a signal transduction pathway leading to malignancy.Drugs or antibodies which block the binding site of MN protein forreceptor H would be expected to cause reversion ofprenoplastic/neoplastic cells to normal or induce their death.

Design and Development of MN-Blocking Drugs or Antibodies

A process to design and develop MN-blocking drugs, e.g., peptides withhigh affinity to MN protein, or antibodies, has several steps. First, isto test for the binding of MN protein to receptors based on the celladhesion assay described infra. That same procedure would also be usedto assay for drugs blocking the MN protein binding site. In view of thealternative receptors S and H, stomach epithelial cells or revertants(containing preferentially S receptors), HeLa cells (containing the Hreceptor and lacking the S receptor) would be used in the cell adhesionassay.

To identify the receptor binding site of MN protein, deletion variantsof MN protein lacking different domains can be used to identifyregion(s) responsible for interaction of MN protein with a receptor.Example 4 has identified MN's binding site as closely related oridentical to the epitope for MAb M75, which is located in at least 2copies within the 6-fold tandem repeat of 6 amino acids [aa 61-96 (SEQID NO: 97)] in the proteoglycan domain of the MN protein. Smallerdeletion variants can be prepared within that relevant domain, e.g.,fusion proteins with only small segments of MN protein can be prepared.Also, controlled digestion of MN protein with specific proteasesfollowed by separation of the products can be performed.

Further, peptides comprising the expected binding site can besynthesized. All of those products can be tested in cell adhesionassays, as exemplified below. [See, e.g., Pierschbacher and Ruoslahti,PNAS, 81:5985 (1984); Ruoslahti and Pierschbacher, Science. 238: 491.]

Molecules can be constructed to block the MN receptor binding site. Forexample, use of a phage display peptide library kit [as Ph.D®-7 Peptide7-Mer Library Kit from New England Biolabs; Beverly, Mass. (USA)] asexemplified in Example 5, can be used to find peptides with highaffinity to the target molecules. Biologic activity of the identifiedpeptides will be tested in vitro by inhibition of cell adhesion to MNprotein, by effects on cell morphology and growth characteristics ofMN-related tumor cells (HeLa) and of control cells. [Symington, J. Biol.Chem., 267: 25744 (1992).] In vivo screening will be carried out in nudemice that have been injected with HeLa cells.

Peptides containing the binding site of the MN protein will be prepared[e.g. MAPs (multiple antigen peptides); Tam, J. P., PNAS (USA) 85: 5409(1988); Butz et al., Peptide Res., 7: 20 (1994)]. The MAPs will be usedto immunize animals to obtain antibodies (polyclonal and/or monoclonal)that recognize and block the binding site. [See, e.g., Brooks et al.,Cell, 79: 1157 (1994).] “Vaccination” would then be used to test forprotection in animals. Antibodies to the MN binding site couldpotentially be used to block MN protein's interaction(s) with othermolecules.

Computer modeling can also be used to design molecules with specificaffinity to MN protein that would mediate steric inhibition between MNprotein and its receptor. A computer model of the MN binding site forthe receptor will contain spatial, electrostatic, hydrophobic and othercharacteristics of this structure. Organic molecules complementary tothe structure, that best fit into the binding site, will be designed.

The use of oncoproteins as targets for developing new cancertherapeutics is considered conventional by those of skill in the art.[See, e.g., Mendelsohn and Lippman, “Growth Factors,” pp. 114-133, IN:DeVita et al. (eds.), Cancer: Principles and Practice of Oncology(4^(th) Ed.; Lippincott; Philadelphia, 1993).] In its broadest sense,the design of blocking drugs can be based in competitive inhibitionexperiments. Such experiments have been used to invent drugs since thediscovery of sulfonamides (competitive inhibitors of para-aminobenzoicacid, a precursor of folic acid). Also, some cytostatics are competitiveinhibitors (e.g., halogenated pyrimidines, among others).

However, the application of such approaches to MN is new. In comparisonto other tumor-related molecules (e.g. growth factors and theirreceptors), MN has the unique property of being differentially expressedin preneoplastic/neoplastic and normal tissues, which are separated byan anatomic barrier.

MN Gene—Cloning and Sequencing

FIG. 1A-C provides the nucleotide sequence for a full-length MN cDNAclone isolated as described below [SEQ ID NO: 1]. FIG. 2A-F provides acomplete MN genomic sequence [SEQ ID NO: 5]. FIG. 6 shows the nucleotidesequence for a proposed MN promoter [SEQ ID NO: 27].

It is understood that because of the degeneracy of the genetic code,that is, that more than one codon will code for one amino acid [forexample, the codons TTA, TTG, CTT, CTC, CTA and CTG each code for theamino acid leucine (leu)], that variations of the nucleotide sequencesin, for example, SEQ ID NOS: 1 and 5 wherein one codon is substitutedfor another, would produce a substantially equivalent protein orpolypeptide according to this invention. All such variations in thenucleotide sequences of the MN cDNA and complementary nucleic acidsequences are included within the scope of this invention.

It is further understood that the nucleotide sequences herein describedand shown in FIGS. 1, 2 and 6, represent only the precise structures ofthe cDNA, genomic and promoter nucleotide sequences isolated anddescribed herein. It is expected that slightly modified nucleotidesequences will be found or can be modified by techniques known in theart to code for substantially similar or homologous MN proteins andpolypeptides, for example, those having similar epitopes, and suchnucleotide sequences and proteins/polypeptides are considered to beequivalents for the purpose of this invention. DNA or RNA havingequivalent codons is considered within the scope of the invention, asare synthetic nucleic acid sequences that encode proteins/polypeptideshomologous or substantially homologous to MN proteins/polypeptides, aswell as those nucleic acid sequences that would hybridize to saidexemplary sequences [SEQ. ID. NOS. 1, 5 and 27] under stringentconditions, or that, but for the degeneracy of the genetic code wouldhybridize to said cDNA nucleotide sequences under stringenthybridization conditions. Modifications and variations of nucleic acidsequences as indicated herein are considered to result in sequences thatare substantially the same as the exemplary MN sequences and fragmentsthereof.

Partial cDNA Clone

In Zavada et al., id., the isolation of a partial MN cDNA clone of 1397bp in length was described. A lambda gt11 cDNA library of LMCV-infectedHeLa cells was prepared and subjected to immunoscreening with Mab M75 incombination with goat anti-mouse antibodies conjugated with alkalinephosphatase. One positive clone was picked and subcloned into the NotIsite of pBluescript KS [Stratagene; La Jolla, Calif. (USA)] therebycreating pBluescript-MN.

Two oppositely oriented nested deletions were made using ERASE-A-BASE®KIT [set of reagents and protocols for the construction ofunidirectional nested deletion sets from plasmid or M13 clones using theprocedure developed by Henikoff, S., Gene, 28: 351 (1984); Promega;Madison, Wis. (USA)] and sequenced by dideoxy method with a T7sequencing kit [Pharmacia; Piscataway, N.J. (USA)]. The sequencingshowed a partial cDNA clone, the insert being 1397 bp long. The sequencecomprises a large 1290 bp open reading frame and 107 bp 3′ untranslatedregion containing a polyadenylation signal (AATAAA). However, thesequence surrounding the first ATG codon in the open reading frame (ORF)did not fit the definition of a translational start site. In addition,as followed from a comparison of the size of the MN clone with that ofthe corresponding mRNA in a Northern blot, the cDNA was shown to bemissing about 100 bp from the 5′ end of its sequence.

Full-Length cDNA Clone

Attempts to isolate a full-length clone from the original cDNA libraryfailed. Therefore, the inventors performed a rapid amplification of cDNAends (RACE) using MN-specific primers, R1 and R2 [SEQ ID NOS: 7 and 8],derived from the 5′ region of the original cDNA clone. The RACE productwas inserted into pBluescript, and the entire population of recombinantplasmids was sequenced with an MN-specific primer ODN1 [SEQ ID NO: 3].In that way, a reliable sequence at the very 5′ end of the MN cDNA asshown in FIG. 1 [SEQ ID NO: 1] was obtained.

Specifically, RACE was performed using 5′ RACE System [GIBCO BRL;Gaithersburg, Md. (USA)] as follows. 1 μg of mRNA (the same as above)was used as a template for the first strand cDNA synthesis which wasprimed by the MN-specific antisense oligonucleotide, R1(5′-TGGGGTTCTTGAGGATCTCCAGGAG-3′) [SEQ ID NO: 7]. The first strandproduct was precipitated twice in the presence of ammonium acetate and ahomopolymeric C tail was attached to its 3′ end by TdT. Tailed cDNA wasthen amplified by PCR using a nested primer, R2(5′-CTCTMCTTCAGGGAGCCCTCTTCTT-3′) [SEQ ID NO: 8] and an anchor primerthat anneals to the homopolymeric tail (5′-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGI IGGGIIGGGIIG-3′) [SEQ ID NO: 9]. The amplified product wasdigested with BamHI and SalI restriction enzymes and cloned intopBluescript II KS plasmid. After transformation, plasmid DNA waspurified from the whole population of transformed cells and used as atemplate for sequencing with the MN-specific primer ODN1 [SEQ ID NO: 3;a 29-mer 5′ CGCCCAGTGGGTCATCTTCCCCAGAAGAG 3′].

Based upon results of the RACE analysis, the full-length MN cDNAsequence was seen to contain a single ORF starting at position 12, withan ATG codon that is in a good context (GCGCATGG) with the rule proposedfor translation initiation [Kozak, J. Cell. Biol., 108: 229-241 (1989)].The AT rich 3′ untranslated region contains a polyadenylation signal(AATAAA) preceding the end of the cDNA by 10 bp. Surprisingly, thesequence from the original clone as well as from four additional clonesobtained from the same cDNA library did not reveal any poly(A) tail.Moreover, just downstream of the poly(A) signal, an ATTTA motif that isthought to contribute to mRNA instability [Shaw and Kamen, Cell, 46:659-667 (1986)] was found. That fact raised the possibility that thepoly(A) tail is missing due to the specific degradation of the MN mRNA.

Genomic Clones

To study MN regulation, MN genomic clones were isolated. One MN genomicclone (Bd3) was isolated from a human cosmid library prepared from fetalbrain using both MN cDNA as a probe and the MN-specific primers derivedfrom the 5′ end of the cDNA ODN1 [SEQ ID NO: 3, supra] and ODN2 [SEQ. IDNO.: 4; 19-mer (5′ GGMTCCTCCTGCATCCGG 3′)]. Sequence analysis revealedthat that genomic clone covered a region upstream from a MNtranscription start site and ending with the BamHI restriction sitelocalized inside the MN cDNA. Other MN genomic clones can be similarlyisolated.

In order to identify the complete genomic region of MN, the humangenomic library in Lambda FIX II vector (Stratagene) was prepared fromHeLa chromosomal DNA and screened by plaque hybridization using MN cDNAas described below. Several independent MN recombinant phages wereidentified, isolated and characterized by restriction mapping andhybridization analyses. Four overlapping recombinants covering the wholegenomic region of MN were selected, digested and subcloned intopBluescript. The subclones were then subjected to bidirectional nesteddeletions and sequencing. DNA sequences were compiled and analyzed bycomputer using the DNASIS software package.

FIG. 7 provides a schematic of the alignment of MN genomic clonesaccording to the transcription initiation site. Plasmids containing theA4a clone and the XE1 and XE3 subclones were deposited at the AmericanType Culture Collection (ATCC) on Jun. 6, 1995, respectively under ATCCDeposit Nos. 97199, 97200, and 97198.

Exon-Intron Structure of Complete MN Genomic Region

The complete sequence of the overlapping clones contains 10,898 bp (SEQID NO: 5). FIG. 5 depicts the organization of the human MN gene, showingthe location of all 11 exons as well as the 2 upstream and 6 intronicAlu repeat elements. All the exons are small, ranging from 27 to 191 bp,with the exception of the first exon which is 445 bp. The intron sizesrange from 89 to 1400 bp.

Table 1 below lists the splice donor and acceptor sequences that conformto consensus splice sequences including the AG-GT motif [Mount, NucleicAcids Res. 10: 459-472 (1982)]. TABLE 1 Exon-Intron Structure of theHuman MN Gene SEQ SEQ Genomic ID 5′splice ID Exon Size Position** NOdonor NO 1 445 *3507-3951  28 AGAAG gtaagt 67 2 30 5126-5155 29 TGGAGgtgaga 68 3 171 5349-5519 30 CAGTC gtgagg 69 4 143 5651-5793 31 CCGAGgtgagc 70 5 93 5883-5975 32 TGGAG gtacca 71 6 67 7376-7442 33 GGAAGgtcagt 72 7 158 8777-8934 34 AGCAG gtgggc 73 8 145 9447-9591 35 GCCAGgtacag 74 9 27 9706-9732 36 TGCTG gtgagt 75 10 82 10350-10431 37 CACAGgtatta 76 11 191 10562-10752 38 ATAAT end SEQ SEQ Genomic ID 3′splice IDIntron Size Position ** NO acceptor NO 1 1174 3952-5125 39 atacag GGGAT77 2 193 5156-5348 40 ccccag GCGAC 78 3 131 5520-5650 41 acgcag TGCAA 794 89 5794-5882 42 tttcag ATCCA 80 5 1400 5976-7375 43 ccccag GAGGG 81 61334 7443-8776 44 tcacag GCTCA 82 7 512 8935-9446 45 ccctag CTCCA 83 8114 9592-9705 46 ctccag TCCAG 84 9 617 9733-10349 47 tcgcag GTGACA 85 10130 10432-10561 48 acacag AAGGG 86** positions are related to nt numbering in whole genomic sequenceincluding the 5′flanking region [FIG. 2A-F]* number corresponds to transcription initiation site determined belowby RNase protection assay

The CA domain is encoded by exons 2-8, while the exons 1, 10 and 11correspond respectively to the proteoglycan-like domain, thetransmembrane anchor and cytoplasmic tail of the MN/CA IX protein. Thatstructure suggests that MN is a chimeric gene assembled by exonshuffling. The gene diverged from an ancestral CA gene at a relativelyearly stage of evolution as indicated by the intron distribution in theCA domain coding region as well as by amino acid sequence homology.

The exon/intron pattern of CA coding region of MN/CA IX is closer tothat of CA IV gene that encodes a membrane-associated isoenzyme than ofgenes corresponding to cytoplasmic and mitochondrial isoenzymes.However, based on amino acid sequence homology, MN/CA IX is more closelyrelated to the secreted CA VI than to any other isoenzyme. Since CA IV,CA VI and CARP/CA VIII are thought to have diverged the earliest, CA VIIand CA V somewhat later and CAs I, II, III more recently [Tashian, R.E., Adv. in Genetics, 30: 321-356 (1992)], it appears that MN/CA IXbelongs to the oldest mammalian CA genes.

The structure of the genomic region coding for the CA domain of MN/CA IXis similar in possessing seven exons, to other CA genes characterized sofar, except for CA I and CA IV genes that have an additional exon attheir 5′ end [Lowe et al., Gene, 93: 277-283 (1990); Okuyama et al.,Genomics, 16: 678-684 (1993).] In the cytosolic and mitochondrial CAgenes (CA I, II, III, V and VII, six introns are distributed atidentical positions. The CA IV gene, encoding the membrane-boundisoenzyme, shares positions of only three 3′ introns (introns 3, 4 and6) with the cytosolic and mitochondrial CAs. The fourth position (intron5) shows a 1 bp slippage at both the donor and acceptor sites. Theremaining introns involve either separate insertion events orconsiderable slippage with little or no change in polypeptide length.Alignment of human CAs, whose gene structure has already beendetermined, shows with respect to the placement of introns 2 and 5,MN/CA IX is more similar to CA IV, than to CA I, II, III, V, and VII.However, the position of intron 1 appears closer to that of CA I, II,III, V and VII. Generally, the coding sequence of the CA domain of theMN/CA IX protein is divided in a manner analogous to sequences of otherCA genes. That fact supports the view that MN/CA IX belongs to the CAgene family.

Mapping of MN Gene Transcription Initiation Site

In the earlier attempt to localize the site of transcription initiationof the MN gene by RACE (above), obtained was a major PCR fragment whosesequence placed the start site 12 bp upstream from the first codon ofthe ORF. That result was obtained probably due to a preferentialamplification of the shortest form of mRNA. Therefore, the inventorsused an RNase protection assay (RNP) for fine mapping of the 5′ end ofthe MN gene. The probe was a uniformly labeled 470 nucleotide copy RNA(nt −205 to +265) [SEQ ID NO: 55], which was hybridized to total RNAfrom MN-expressing HeLa and CGL3 cells and analyzed on a sequencing gel.That analysis has shown that the MN gene transcription initiates atmultiple sites, the 5′ end of the longest MN transcript being 30 ntlonger than that previously characterized by RACE.

Mapping of MN Gene Transcription Termination Site

An RNase protection assay was used to verify the 3′ end of the MN cDNA.That was important with respect to our previous finding that the cDNAcontains a poly(A) signal but lacks a poly(A) tail, which could be lostduring the proposed degradation of MN mRNA due to the presence of aninstability motif in its 3′ untranslated region. RNP analysis of MN mRNAwith the fragment of the genomic clone XE3 covering the region ofinterest corroborated our data from MN cDNA sequencing, since the 3′ endof the protected fragment corresponded to the last base of MN cDNA(position 10,752 of the genomic sequence). That site also meets therequirement for the presence of a second signal in the genomic sequencethat is needed for transcription termination and polyadenylation[McLauchlan et al., Nucleic Acids Res., 13: 1347 (1985)]. MotifTGTGTTAGT (nt 10,759-10,767) corresponds well to both the consensussequence and the position of that signal within 22 bp downstream fromthe polyA signal (nt 10,737-10,742).

Characterization of the 5′ Flanking Region

The Bd3 genomic clone isolated from human fetal brain cosmid library wasfound to cover a region of 3.5 kb upstream from the transcription startsite of the MN gene. It contains no significant coding region. Two Alurepeats are situated at positions −2587 to −2296 [SEQ ID NO: 56] and−1138 to −877 [SEQ ID NO: 57] (with respect to the transcription startdetermined by RNP). The sequence proximal to the 5′ end is stronglyhomologous (91.4% identity) to the U3 region of long terminal repeats ofhuman endogenous retroviruses HERV-K [Ono, M., J. Virol. 58: 937-944(1986)]. The LTR-like fragment is 222 bp long with an A-rich tail at its3′ end. Most probably, it represents part of SINE (short interspersedrepeated sequence) type nonviral retroposon derived from HERV-K [Ono etal., Nucleic Acids Res., 15: 8725-8373 (1987)]. There are no sequencescorresponding to regulatory elements in this fragment, since the 3′ partof U3, and the entire R and U5 regions of LTR are absent from the Bd3genomic clone, and the glucocorticoid responsive element as well as theenhancer core sequences are beyond its 5′ border.

However, two keratinocyte-dependent enhancers were identified in thesequence downstream from the LTR-like fragment at positions −3010 and−2814. Those elements are involved in transcriptional regulation of theE6-E7 oncogenes of human papillomaviruses and are thought to account fortheir tissue specificity [Cripe et al., EMBO J., 6: 3745-3753 (1987)].

Nucleotide sequence analysis of the DNA 5′ to the transcription start(from nt −507) revealed no recognizable TATA box within the expecteddistance from the beginning of the first exon. However, the presence ofpotential binding sites for transcription factors suggests that thisregion might contain a promoter for the MN gene. There are severalconsensus sequences for transcription factors AP1 and AP2 as well as forother regulatory elements, including a p53 binding site [Locker andBuzard, J., DNA Sequencing and Mapping, 1: 3-11 (1990); Imagawa et al.Cell, 51: 251-260 (1987); El Deiry et al., Nat. Genet., 1: 44-49(1992)]. Although the putative promoter region contains 59.3% C+G, itdoes not have additional attributes of CpG-rich islands that are typicalfor TATA-less promoters of housekeeping genes [Bird, Nature, 321:209-213 (1986)]. Another class of genes lacking TATA box utilizes theinitiator (Inr) element as a promoter. Many of these genes are notconstitutively active, but they are rather regulated duringdifferentiation or development. The Inr has a consensus sequence ofPyPyPyCAPyPyPyPyPy [SEQ ID NO: 23] and encompasses the transcriptionstart site [Smale and Baltimore, Cell, 57: 103-113 (1989)]. There aretwo such consensus sequences in the MN putative promoter; however, theydo not overlap the transcription start (FIG. 6).

An interesting region was found in the middle of the MN gene. The regionis about 1.4 kb in length [nt 4,600-6,000 of the genomic sequence; SEQID NO: 49] and spans from the 3′ part of the 1 st intron to the end ofthe 5th exon. The region has the character of a typical CpG-rich island,with 62.8% C+G content and 82 CpG: 131 GpC dinucleotides. Moreover,there are multiple putative binding sites for transcription factors AP2and Sp1 [Locker and Buzard, supra; Briggs et al., Science. 234: 47-52(1986)] concentrated in the center of this area. Particularly the 3rdintron of 131 bp in length contains three Sp1 and three AP2 consensussequences. That data indicates the possible involvement of that regionin the regulation of MN gene expression. However, functionality of thatregion, as well as other regulatory elements found in the proposed 5′ MNpromoter, remains to be determined.

MN Promoter

Study of the MN promoter has shown that it is TATA-less and containsregulatory sequences for AP-1, AP-2, as well as two p53 binding sites.The sequence of the 5′ end of the 3.5 kb flanking region upstream of theMN gene has shown extensive homology to LTR of HERV-K endogenousretroviruses. Basal transcription activity of the promoter is very weakas proven by analyses using CAT and neo reporter genes. However,expression of the reporter genes is severalfold increased when drivenfrom the 3.5 kb flanking region, indicating involvement of putativeenhancers.

Utility of MN Promoter as a Tumor-Specific Promoter for Gene Therapy

Being investigated is whether the MN gene promoter can be used as atumor-specific promoter to drive the expression of a suicide gene[thymidine kinase (tk) of HSV)] and mediate the direct and bystanderkilling of tumor cells. HSVtk gene transferred to tumor cells convertsnucleoside analogue ganciclovir (GCV) to toxic triphophates and mediatesthe death of transduced and also neighboring tumor cells. The control ofHSVtk by the MN gene promoter would allow its expression only in tumorcells, which are permissive for the biosynthesis of MN protein, andselectively kill such tumor cells, but not normal cells in which MNexpression is repressed.

A plasmid construct in which HSVtk was cloned downstream of the MNpromoter region Bd3, containing both proximal and distant regulatoryelements of MN, was prepared. That plasmid pMN-HSVtk was transfected toRat2TK-cells and C33 human cervical carcinoma cells using calciumphosphate precipitation and lipofection, respectively. Transfectantswere tested for expression of HSVtk and GVC sensitivity. Analysis of thetransfectants has shown the remarkable cytotoxic in vitro effect of GVCeven in low concentrations (up to 95% of cells killed).

Polyclonal rabbit antiserum against HSVtk, using fusion protein with GSTin pGEX-3×, has been prepared to immunodetect HSVtk synthesized intransfected cells. This model system is being studied to estimate thebystander effect, the inhibition of cloning efficiency and invasivenessof transduced and GVC-treated cells to collagen matrices. A recombinantretroviral vector with the MN promoter-driven HSVtk is to be prepared totest its in vivo efficacy using an animal model (e.g., SCID-mouse).

MN Promoter Analysis

Since the MN promoter is weak, a classical approach to study it would belimited due to the relatively low efficiency of transient transfections(up to 10%). Therefore, stable clonal cell lines expressing constructscontaining the MN promoter fused to the CAT gene were prepared. In suchclonal lines, 100% of the cells express the CAT gene driven from the MNpromoter, and thus, the activity of the promoter is detectable easierthan in transient experiments. Also, the promoter activity can beanalysed repeatedly in the same cells under different conditions ortreated by different factors and drugs. This approach allows for thestudy of the mechanisms underlying MN regulation at the level oftranscription initiation.

Several types of transfections with promoter constructs linked to areporter CAT gene (calcium precipitation, DEAE dextran combined withDMSO shock and/or chloroquine, as well as electroporation), differentmethods of CAT activity assay (scintillation method, thin layerchromatography) and several recipient cell lines differing in the levelof MN expression and in transfection efficiency (HeLa, SiHa, CGL3, KATOIII, Rat2TK⁻ and C33 cells). Activity of the MN promoter was detectedpreferably by the electroporation of CGL3 cells and thin layerchromatography. Further preferably, C33 cells cotransfected with MNpromoter-CAT constructs and pSV2neo were used.

1. To detect basal activity of the MN promoter and to estimate theposition of the core promoter, expression of the CAT gene fromconstructs pMN1 to pMN7 after transfection to CGL3 cells was analyzed.Plasmids with progressive 5′ deletions were transfected into CGL3 cellsand activity was analyzed by CAT assay. [8 μg of DNA was used fortransfection in all cases except pBLV-LTR (2 μg).]

Only very weak CAT activity was detected in cells transfected by pMN1and pMN2 (containing respectively 933 bp and 600 bp of the promotersequence). A little higher activity was exhibited with the constructspMN3, pMN4 and pMN6 (containing respectively 446 bp, 243 bp and 58 bp ofthe promoter). A slight peak of activity was obtained with pMN5(starting at position −172 with respect to the transcription start.)Thus, the function of the MN core promoter can be assigned to a regionof approximately 500 bp immediately upstream from the MN transcriptioninitiation site.

Interestingly, the activity of the large Bd3 region (covering 3.5 kbpupstream of the transcription start) was severalfold higher than theactivity of the core promoter. However, its level was still much lowerthan that exhibited by a positive control, i.e., BLV-LTR transactivatedby Tax, and even lower than the activity of BLV-LTR withouttransactivation. That the activity of Bd3 was elevated in comparison tothe core promoter suggests the presence of some regulatory elements.Such elements are most probably situated in the sequence between pMN1and Bd3 (i.e. from −1 kbp to −3.5 kbp) [SEQ ID NO: 58]. The cloning andtransfection of several deletion versions of Bd3 covering the indicatedregion can be used to determine the location of the putative regulatoryelements.

Similar results were obtained from transfecting KATO III cells with Bd3and pMN4. The transfected cells expressed a lower level of MN than theCGL3 cells. Accordingly, the activity of the MN promoter was found to belower than in CGL3 cells.

2. In a parallel approach to study the MN promoter, an analysis based onG418 selection of cells transfected by plasmids containing the promoterof interest cloned upstream from the neo gene was made. This approach issuitable to study weak promoters, since its sensitivity is much higherthan that of a standard CAT assay. The principle underlying the methodis as follows: an active promoter drives expression of the neo genewhich protects transfected cells from the toxic effect of G418, whereasan inactive promoter results in no neo product being made and the cellstransfected thereby die upon the action of G418. Therefore, the activityof the promoter can be estimated according to the number of cellcolonies obtained after two weeks of selection with G418. Threeconstructs were used in the initial experiments—pMN1neo, pMN4neo andpMN7neo. As pMN7neo contains only 30 bp upstream of the transcriptionstart site, it was considered a negative control. As a positive control,pSV2neo with a promoter derived from SV40 was used. Rat2TK⁻ cells werechosen as the recipient cells, since they are transfectable with highefficiency by the calcium precipitation method.

After transfection, the cells were subjected to two weeks of selection.Then the medium was removed, the cells were rinsed with PBS, and thecolonies were rendered visible by staining with methylene blue. Theresults obtained from three independent experiments corroborated thedata from the CAT assays. The promoter construct pMN4neo exhibitedhigher transcriptional activity than pMN1neo. However, the differencebetween the positive control and pMN4neo was not so striking as in theCAT assay. That may have been due to both lower promoter activity ofpSV2neo compared to Tax-transactivated pBLV-LTR and to differentconditions for cell growth after transfection. From that point of view,stable transfection is probably more advantageous for MN expression,since the cells grow in colonies with close cell to cell contact, andthe experiment lasts much longer, providing a better opportunity todetect promoter activity.

3. Stable transfectants expressing MN promoter-CAT chimeric genes wereprepared by the cotransfection of relevant plasmids with pSV2neo. Asrecipient cells, HeLa cells were used first. However, no clonesexpressing the promoter-CAT constructs were obtained. That negativeresult was probably caused by homologic recombination of the transfectedgenomic region of MN (e.g. the promoter) with the correspondingendogenous sequence. On the basis of that experience, C33 cells derivedfrom a HPV-negative cervical carcinoma were used. C33 cells do notexpress MN, since during the process of tumorigenesis, they lost geneticmaterial including chromosomal region 9p which contains the MN gene. Inthese experiments, the absence of the MN gene may represent an advantageas the possibility of homologic recombinations is avoided.

C33 Cells Transfected with MN Promoter-CAT Constructs

C33 cells expressing the CAT gene under MN promoter regions Bd3(−3500/+31) [SEQ ID NO: 90] and MN5 (−172/+31) [SEQ ID NO: 91] were usedfor initial experiments to analyze the influence of cell density on thetranscriptional activity of the MN promoter. The results indicated thatsignals generated after cells come into close contact activatetranscription of the CAT protein from the MN promoter in proportion tothe density of the cell culture. Interestingly, the data indicated thatthe MN protein is not required for this phase of signal transduction,since the influence of density is clearly demonstrated in MN-negativeC33 cells. Rather, it appears that MN protein acts as an effectormolecule produced in dense cells in order to perform a certainbiological function (i.e., to perturb contact inhibition). Alsointerestingly, the MN promoter activity is detectable even in verysparse cell cultures suggesting that MN is expressed at a very low levelalso is sparse subconfluent culture.

Deletion Variants. Deletion variants of the Bd3-CAT promoter constructwere then prepared. The constructs were cotransfected with pSV2neo intoC33 cervical cells. After selection with G418, the whole population ofstably transfected cells were subjected to CAT ELISA analysis.Expression of the deletion constructs resulted in the synthesis ofsimilar levels of CAT protein to that obtained with the Bd3-CATconstruct. On the basis of that preliminary data, the inventors proposedthat sequences stimulating transcription of MN are located between −3506and −3375 bp [SEQ ID NO: 92] upstream from the transcription start. Thatis the sequence exhibiting homology to HERV-K LTR.

However, transient transfection studies in CGL3 cells repeatedlyrevealed that the LTR region is not required for the enhancement ofbasal MN promoter activity. Further, results obtained in CGL3 cellsindicate that the activating element is localized in the region from−933 to −2179 [SEQ ID NO: 110] with respect to transcription initiationsite (the position of the region having been deduced from overlappingsequences in the Bd3 deletion mutants).

Interaction of Nuclear Proteins with MN Promoter Sequences

In order to identify transcription factors binding to the MN promoterand potentially regulating its activity, a series of analyses using anelectrophoretic mobility shift assay (EMSA) and DNase I footprintinganalysis (FTP) were performed.

EMSA

In the EMSA, purified promoter fragments MN4 (−243/+31) [SEQ ID NO: 93],MN5 (−172/+31) [SEQ ID NO: 91], MN6 (−58/+31) [SEQ ID NO: 94] and pMN7(−30/+31) [SEQ ID NO: 95], labeled at the 3′ ends by Klenow enzyme, wereallowed to interact with proteins in nuclear extracts prepared from CGL1and CGL3 cells. [40 μg of nuclear proteins were incubated with 30,000cpm end-labeled DNA fragments in the presence of 2 μg poly(dIdC).]DNA-protein complexes were analysed by PAGE (native 6%), where thecomplexes created extra bands that migrated more slowly than the freeDNA fragments, due to the shift in mobility which is dependent on themoiety of bound protein.

The EMSA of the MN4 and MN5 promoter fragments revealed severalDNA-protein complexes; however, the binding patterns obtainedrespectively with CGL1 and CGL3 nuclear extracts were not identical.There is a single CGL-1 specific complex.

The EMSA of the MN6 promoter fragment resulted in the formation of threeidentical complexes with both CGL1 and CGL3 nuclear extracts, whereasthe MN7 promoter fragment did not bind any nuclear proteins.

The EMSA results indicated that the CGL1 nuclear extract contains aspecific factor, which could participate in the negative regulation ofMN expression in CGL1 cells. Since the specific DNA-protein complex isformed with MN4 (−243/+31) [SEQ. ID NO.: 93] and MN5 (−172/+31) [SEQ. IDNO.: 91] promoter fragments, but not with MN6 (−58/+31) [SEQ ID NO: 94],it appears that the binding site of the protein component of thatspecific complex is located between −173 and −58 bp [SEQ. ID NO.: 96]with respect to transcription initiation.

The next step was a series of EMSA analyses using double stranded (ds)oligonucleotides designed according to the protected regions in FTPanalysis. A ds oligonucleotide derived from the protected region PR2[covering the sequence from −72 to −56 bp (SEQ ID NO: 111)] of the MNpromoter provided confirmation of the binding of the AP-1 transcriptionfactor in competitive EMSA using commercial ds olignucleotidesrepresenting the binding site for AP-1.

EMSA of ds oligonucleotides derived from the protected regions of PR1[−46 to −24 bp (SEQ ID NO: 112)], PR2 [−72 to −56 bp (SEQ ID NO: 111)],PR3 [−102 to −85 (SEQ ID NO: 113)] and PR5 [−163 to −144 (SEQ ID NO:114)] did not reveal any differences in the binding pattern of nuclearproteins extracted from CGL1 and CGL3 cells, indicating that thoseregions do not bind crucial transcription factors which controlactivation of the MN gene in CGL3, or its negative regulation in CGL1.However, EMSA of ds oligonucleotides from the protected region PR4 [-133to −108; SEQ ID NO: 115] repeatedly showed remarkable quantitativedifferences between binding of CGL1 and CGL3 nuclear proteins. CGL1nuclear proteins formed a substantially higher amount of DNA-proteincomplexes, indicating that the PR4 region contains a binding site forspecific transcription factor(s) that may represent a negative regulatorof MN gene transcription in CGL1 cells. That fact is in accord with theprevious EMSA data which showed CGL-1 specific DNA-protein complex withthe promoter fragments pMN4 (−243/+31; SEQ ID NO: 93) and pMN5(−172/+31; SEQ ID NO: 91), but not with pMN6 (−58/+31; SEQ ID NO: 94).

To identify the protein involved or the formation of a specific complexwith the MN promoter in the PR4 region, relevant ds oligonucleotidescovalently bound to magnetic beads will be used to purify thecorresponding transcription factor. Alternatively the ONE Hybrid System®[Clontech (Palo Alto, Calif. (USA); an in vivo yeast genetic assay forisolating genes encoding proteins that bind to a target, cis-actingregulatory element or any other short DNA-binding sequence] will be usedto search for and clone transcription factors involved in regulation ofthe analysed promoter region. A cDNA library from HeLa cells will beused for that investigation.

FTP

To determine the precise location of cis regulatory elements thatparticipate in the transcriptional regulation of the MN gene, FTP wasused. Proteins in nuclear extracts prepared respectively from CGL1 andCGL3 cells were allowed to interact with a purified ds DNA fragment ofthe MN promoter (MN4, −243/+31) [SEQ ID NO: 93] which was labeled at the5′ end of one strand. [MN4 fragments were labeled either at XhoI site(−243/+31*) or at XbaI site (*−243/+31).] The DNA-protein complex wasthen subjected to DNase I attack, which causes the DNA chain to break atcertain bases if they are not in contact with proteins. [A control usedBSA instead of DNase.] Examination of the band pattern of the denaturedDNA after gel electrophoresis [8% denaturing gel] indicates which of thebases on the labeled strand were protected by protein.

FTP analysis of the MN4 promoter fragment revealed 5 regions (I-V)protected at both the coding and noncoding strand, as well as tworegions (VI and VII) protected at the coding strand but not at thenoncoding strand. FIG. 6 indicates the general regions on the MNpromoter that were protected.

The sequences of the identified protected regions (PR) were subjected tocomputer analysis using the SIGNALSCAN program to see if theycorresponded to known consensus sequences for transcription factors. Thedata obtained by that computer analyses are as follows: PR I codingstrand - AP-2, p53, GAL4 noncoding strand - JCV-repeated PR II codingstrand - AP-1, CGN4 noncoding strand - TCF-1, dFRA, CGN4 PR III codingstrand - no known consensus sequence, only partial overlap of AP1noncoding strand - 2 TCF-1 sites PR IV coding strand - TCF-1, ADR-1noncoding strand - CTCF, LF-A1, LBP-1 PR V coding strand - no knownconsensus motif noncoding strand - JCV repeated PR VI coding strand - noknown consensus motif noncoding strand - T antigen of SV 40, GAL4 PR VIIcoding strand - NF-uE4, U2snRNA.2 noncoding strand - AP-2, IgHC.12,MyoD.

In contrast to EMSA, the FTP analysis did not find any differencesbetween CGL1 and CGL3 nuclear extracts. However, the presence ofspecific DNA-protein interactions detected in the CGL1 nuclear extractsby EMSA could have resulted from the binding of additional protein toform DNA protein-protein complex. If that specific protein did notcontact the DNA sequence directly, its presence would not be detectableby FTP.

EMSA Supershift Analysis

The results of the FTP suggests that transcription factors AP-1, AP-2 aswell as tumor suppressor protein p53 are potentially involved in theregulation of MN expression. To confirm binding of those particularproteins to the MN promoter, a supershift analysis using antibodiesspecific for those proteins was performed. For this analysis,DNA-protein complexes prepared as described for EMSA were allowed tointeract with MAbs or polyclonal antibodies specific for proteinspotentially included in the complex. The binding of antibody to thecorresponding protein results in an additional shift (supershift) inmobility of the DNA-protein-antibody complex which is PAGE visualized asan additional, more slowly migrating band.

By this method, the binding of AP-2 to the MN promoter was confirmed.However, this method did not evidence binding of the AP-1 transcriptionfactor. It is possible that MN protein binds AP-1-related protein, whichis antigenically different from the AP-1 recognized by the antibodiesused in this assay.

Also of high interest is the possible binding of the p53 tumorsuppressor protein to the MN promoter. It is well known that wt p53functions as a transcription factor, which activates expression ofgrowth-restricting genes and down-modulates, directly or indirectly, theexpression of genes that are required for ongoing cell proliferation.Transient co-transfection experiments using the pMN4-CAT promoterconstruct in combination with wt p53 cDNA and mut p53 cDNA,respectively, suggested that wt p53, but not mut p53, negativelyregulates expression of MN. In addition, one of two p53-binding sites inthe MN promoter is protected in FTP analysis (FIG. 6), indicating thatit binds to the corresponding protein. Therefore, supershift analysis toprove that p53 binds to the MN promoter with two p53-specificantibodies, e.g. Mabs 421 and DO-1 [the latter kindly provided by Dr.Vojtesek from Masaryk Memorial Cancer Institute in Brno, Czech Republic]are to be performed with appropriate nuclear extracts, e.g. from MCF-7breast carcinoma cells which express wt p53 at a sufficient level.

Regulation of MN Expression and MN Promoter

MN appears to be a novel regulatory protein that is directly involved inthe control of cell proliferation and in cellular transformation. InHeLa cells, the expression of MN is positively regulated by celldensity. Its level is increased by persistent infection with LCMV. Inhybrid cells between HeLa and normal fibroblasts, MN expressioncorrelates with tumorigenicity. The fact that MN is not present innontumorigenic hybrid cells (CGL1), but is expressed in a tumorigenicsegregant lacking chromosome 11, indicates that MN is negativelyregulated by a putative suppressor in chromosome 11.

Evidence supporting the regulatory role of MN protein was found in thegeneration of stable transfectants of NIH 3T3 cells that constitutivelyexpress MN protein. As a consequence of MN expression, the NIH 3T3 cellsacquired features associated with a transformed phenotype: alteredmorphology, increased saturation density, proliferative advantage inserum-reduced media, enhanced DNA synthesis and capacity foranchorage-independent growth. Further, flow cytometric analyses ofasynchronous cell populations indicated that the expression of MNprotein leads to accelerated progression of cells through G1 phase,reduction of cell size and the loss of capacity for growth arrest underinappropriate conditions. Also, MN expressing cells display a decreasedsensitivity to the DNA damaging drug mitomycin C.

Nontumorigenic human cells, CGL1 cells, were also transfected with thefull-length MN cDNA. The same pSG5C-MN construct in combination withpSV2neo plasmid as used to transfect the NIH 3T3 cells was used. Out of15 MN-positive clones (tested by SP-RIA and Western blotting), 3 werechosen for further analysis. Two MN-negative clones isolated from CGL1cells transfected with empty plasmid were added as controls. Initialanalysis indicates that the morphology and growth habits ofMN-transfected CGL1 cells are not changed dramatically, but theirproliferation rate and plating efficiency is increased.

MN Promoter—Sense/Antisense Constructs

When the promoter region from the MN genomic clone, isolated asdescribed above, was linked to MN cDNA and transfected into CGL1 hybridcells, expression of MN protein was detectable immediately afterselection. However, then it gradually ceased, indicating thus an actionof a feedback regulator. The putative regulatory element appeared to beacting via the MN promoter, because when the full-length cDNA (notcontaining the promoter) was used for transfection, no similar effectwas observed.

An “antisense” MN cDNA/MN promoter construct was used to transfect CGL3cells. The effect was the opposite of that of the CGL1 cells transfectedwith the “sense” construct. Whereas the transfected CGL1 cells formedcolonies several times larger than the control CGL1, the transfectedCGL3 cells formed colonies much smaller than the control CGL3 cells. Thesame result was obtained by antisense MN cDNA transfection in SiHa andHeLa cells.

For those experiments, the part of the promoter region that was linkedto the MN cDNA through a BamHI site was derived from a NcoI-BamHIfragment of the MN genomic clone [Bd3] and represents a region a fewhundred bp upstream from the transcription initiation site. After theligation, the joint DNA was inserted into a pBK-CMV expression vector[Stratagene]. The required orientation of the inserted sequence wasensured by directional cloning and subsequently verified by restrictionanalysis. The tranfection procedure was the same as used in transfectingthe NIH 3T3 cells, but co-transfection with the pSV2neo plasmid was notnecessary since the neo selection marker was already included in thepBK-CMV vector.

After two weeks of selection in a medium containing G418, remarkabledifferences between the numbers and sizes of the colonies grown wereevident as noted above. Immediately following the selection and cloning,the MN-transfected CGL1 and CGL3 cells were tested by SP-RIA forexpression and repression of MN, respectively. The isolated transfectedCGL1 clones were MN positive (although the level was lower than obtainedwith the full-length cDNA), whereas MN protein was almost absent fromthe transfected CGL3 clones. However, in subsequent passages, theexpression of MN in transfected CGL1 cells started to cease, and wasthen blocked perhaps evidencing a control feedback mechanism.

As a result of the very much lowered proliferation of the transfectedCGL3 cells, it was difficult to expand the majority of cloned cells(according to SP-RIA, those with the lowest levels of MN), and they werelost during passaging. However, some clones overcame that problem andagain expressed MN. It is possible that once those cells reached ahigher quantity, that the level of endogenously produced MN mRNAincreased over the amount of ectopically expressed antisense mRNA.

Identification of Specific Transcription Factors Involved in Control ofMN Expression

Control of MN expression at the transcription level involves regulatoryelements of the MN promoter. Those elements bind transcription factorsthat are responsible for MN activation in tumor cells and/or repressionin normal cells. The identification and isolation of those specifictranscription factors and an understanding of how they regulate MNexpression could result in their therapeutic utility in modulating MNexpression.

EMSA experiments indicate the existence of an MN gene repressor. Usingthe One Hybrid System® [Clontech (Palo Alto, Calif.); an in vivo yeastgenetic assay for isolating genes encoding proteins that bind to atarget, cis-acting regulatory element or any other short DNA-bindingsequence; Fields and Song, Nature, 340: 245 (1989); Wu et al., EMBO J.,13: 4823 (1994)] and subtractive suppressive PCR(SSH). SSH allows thecloning of genes that are differentially expressed under conditionswhich are known to up or down regulate MN expression such as densityversus sparsity of HeLa cells, and suspension versus adherent HeLacells.

In experiments with HPV immobilized cervical cells (HCE 16/3), it wasfound that the regulation of MN expression differs from that in fullytransformed carcinoma cells. For example, glucocorticoid hormones, whichactivate HPV transcription, negatively regulate MN expression in HCE,but stimulate MN in HeLa and SiHa. Further keratinocyte growth factors,which down regulates transcription of HPV oncogenes, stimulates MNexpression in suspension HCE but not in adherent cells.

EGF and insulin are involved in the activation of MN expression in bothimmortalized and carcinoma cells. All the noted facts can be used in thesearch for MN-specific transcription factors and in the modulation of MNexpression for therapeutic purposes.

No Direct Relationship in HeLa Cells Between MN Expression andExpression of HPV E6 or p53

Oncogenic potential of human papillomaviruses (HPV) is related tocapacity of HPV-encoded oncoproteins to bind and inactivate tumorsuppressor proteins. The interaction of p53 with HPV E6 results inaberrant regulation of various cellular genes. As the MN gene, isclosely associated with cervical carcinomas, its possible involvement inregulating pathways driven by p53 and E6 was investigated. Discoveredwas that one of the two p53 consensus sequences present in the MNpromoter participate in DNA-protein interaction, but does not bind p53.It was also found that tetracycline-inducible antisense expression ofHPVI8 E6 in human cervical carcinoma cells (HeLa) resulted in anincreased level of p53 that did not affect the expression of MNproteins. Therefore, it was assumed that at least in HeLa cells, thereis no direct relationship between expression of MN and that of E6 orp53.

Deduced Amino Acid Sequence

The ORF of the MN cDNA shown in FIG. 1 has the coding capacity for a 459amino acid protein with a calculated molecular weight of 49.7 kd. Theoverall amino acid composition of the MN/CA IX protein is rather acidic,and predicted to have a pi of 4.3. Analysis of native MN/CA IX proteinfrom CGL3 cells by two-dimensional electrophoresis followed byimmunoblotting has shown that in agreement with computer prediction, theMN/CA IX is an acidic protein existing in several isoelectric forms withpls ranging from 4.7 to 6.3.

As assessed by amino acid sequence analysis, the deduced primarystructure of the MN protein can be divided into four distinct regions.The initial hydrophobic region of 37 amino acids (aa) corresponds to asignal peptide. The mature protein has an N-terminal or extracellularpart of 377 amino acids [aa 38-414 (SEQ ID NO: 87], a hydrophobictransmembrane segment of 20 amino acids [aa 415-434 (SEQ ID NO: 52)] anda C-terminal region of 25 amino acids [aa 435-459 (SEQ ID NO: 53)].

The extracellular part is composed of two distinct domains: (1) aproteoglycan-like domain [aa 53-111 (SEQ ID NO: 50)]; and (2) a CAdomain, located close to the plasma membrane [aa 135-391 (SEQ ID NO:51)]. [The amino acid numbers are keyed to those of FIG. 1.]

More detailed insight into MN protein primary structure disclosed thepresence of several consensus sequences. One potential N-glycosylationsite was found at position 346 of FIG. 1. That feature, together with apredicted membrane-spanning region are consistent with the results, inwhich MN was shown to be an N-glycosylated protein localized in theplasma membrane. MN protein sequence deduced from cDNA was also found tocontain seven S/TPXX sequence elements [SEQ ID NOS: 25 AND 26] (one ofthem is in the signal peptide) defined by Suzuki, J. Mol. Biol., 207:61-84 (1989) as motifs frequently found in gene regulatory proteins.However, only two of them are composed of the suggested consensus aminoacids.

Experiments have shown that the MN protein is able to bind zinc cations,as shown by affinity chromatography using Zn-charged chelatingsepharose. MN protein immunoprecipitated from HeLa cells by Mab M75 wasfound to have weak catalytic activity of CA. The CA-like domain of MNhas a structural predisposition to serve as a binding site for smallsoluble domains. Thus, MN protein could mediate some kind of signaltransduction.

MN protein from LCMV-infected HeLA cells was shown by using DNAcellulose affinity chromatography to bind to immobilized double-strandedsalmon sperm DNA. The binding activity required both the presence ofzinc cations and the absence of a reducing agent in the binding buffer.

CA Domain Required for Anchorage Independence but for IncreasedProliferation of Transfected NIH 3T3 Fibroblasts

In transfected NIH 3T3 fibroblasts, MN protein induces morphologictransformation, increased proliferation and anchorage independence. Theconsequences of constitutive expression of two MN-truncated variants inNIH 3T3 cells were studied. It was found that the proteoglycan-likeregion is sufficient for the morphological alteration of transfectedcells and displays the growth-promoting activity presumably related toperturbation of contact inhibition.

The CA domain is essential for induction of anchorage independence,whereas the TM anchor and IC tail are dispensable for that biologicaleffect. The MN protein is also capable of causing plasma membraneruffling in the transfected cells and appears to participate in theirattachment to the solid support. The data evince the involvement of MNin the regulation of cell proliferation, adhesion and intercellularcommunication.

Sequence Similarities

Computer analysis of the MN cDNA sequence was carried out using DNASISand PROSIS (Pharmacia Software packages). GenBank, EMBL, ProteinIdentification Resource and SWISS-PROT databases were searched for allpossible sequence similarities. In addition, a search for proteinssharing sequence similarities with MN was performed in the MIPS databankwith the FastA program [Pearson and Lipman, PNAS (USA), 85: 2444(1988)].

The proteoglycan-like domain [aa 53-111 (SEQ ID NO: 50)], which isbetween the signal peptide and the CA domain, shows significant homology(38% identity and 44% positivity) with a keratan sulphate attachmentdomain of a human large aggregating proteoglycan aggrecan [Doege et al.,J. Biol. Chem. 266: 894-902 (1991)].

The CA domain [aa 135-391 (SEQ ID NO: 51)] is spread over 265 aa andshows 38.9% amino acid identity with the human CA VI isoenzyme [Aldredet al., Biochemistry. 30: 569-575 (1991)]. The homology between MN/CA IXand other isoenzymes is as follows: 35.2% with CA II in a 261 aa overlap[Montgomery et al., Nucl. Acids. Res., 15: 4687 (1987)], 31.8% with CA Iin a 261 aa overlap [Barlow et al., Nucl. Acids Res., 15: 2386 (1987)],31.6% with CA IV in a 266 aa overlap [Okuyama et al., PNAS (USA) 89:1315-1319 (1992)], and 30.5% with CA III in a 259 aa overlap [Lloyd etal., Genes. Dev., 1: 594-602 (1987)].

In addition to the CA domain, MN/CA IX has acquired both N-terminal andC-terminal extensions that are unrelated to the other CA isoenzymes. Theamino acid sequence of the C-terminal part, consisting of thetransmembrane anchor and the intracytoplasmic tail, shows no significanthomology to any known protein sequence.

The MN gene was clearly found to be a novel sequence derived from thehuman genome. The overall sequence homology between the cDNA MN sequenceand cDNA sequences encoding different CA isoenzymes is in a homologyrange of 48-50% which is considered by ones in the art to be low.Therefore, the MN cDNA sequence is not closely related to any CA cDNAsequences.

Only very closely related nt sequences having a homology of at least80-90% would hybridize to each other under stringent conditions. Asequence comparison of the MN cDNA sequence shown in FIG. 1 and acorresponding cDNA of the human carbonic anhydrase 11 (CA II) showedthat there are no stretches of identity between the two sequences thatwould be long enough to allow for a segment of the CA II cDNA sequencehaving 25 or more nucleotides to hybridize under stringent hybridizationconditions to the MN cDNA or vice versa.

A search for nt sequences related to MN gene in the EMBL Data Librarydid not reveal any specific homology except for 6 complete and 2 partialAlu-type repeats with homology to Alu sequences ranging from 69.8% to91% [Jurka and Milosavljevic, J. Mol. Evol. 32: 105-121 (1991)]. Belowunder the Characterization of the 5′ Flanking Region, also a 222 bpsequence proximal to the 5′ end of the genomic region is shown to beclosely homologous to a region of the HERV-K LTR.

MN Proteins and/or Polypeptides

The phrase “MN proteins and/or polypeptides” (MN proteins/polypeptides)is herein defined to mean proteins and/or polypeptides encoded by an MNgene or fragments thereof. An exemplary and preferred MN proteinaccording to this invention has the deduced amino acid sequence shown inFIG. 1. Preferred MN proteins/polypeptides are those proteins and/orpolypeptides that have substantial homology with the MN protein shown inFIG. 1. For example, such substantially homologous MNproteins/polypeptides are those that are reactive with the MN-specificantibodies of this invention, preferably the Mabs M75, MN12, MN9 and MN7or their equivalents.

A “polypeptide” or “peptide” is a chain of amino acids covalently boundby peptide linkages and is herein considered to be composed of 50 orless amino acids. A “protein” is herein defined to be a polypeptidecomposed of more than 50 amino acids.

MN proteins exhibit several interesting features: cell membranelocalization, cell density dependent expression in HeLa cells,correlation with the tumorigenic phenotype of HeLa×fibroblast somaticcell hybrids, and expression in several human carcinomas among othertissues. As demonstrated herein, for example, in Example 1, MN proteincan be found directly in tumor tissue sections but not in general incounterpart normal tissues (exceptions noted infra as in normal gastricmucosa and gallbladder tissues). MN is also expressed sometimes inmorphologically normal appearing areas of tissue specimens exhibitingdysplasia and/or malignancy. Taken together, these features suggest apossible involvement of MN in the regulation of cell proliferation,differentiation and/or transformation.

It can be appreciated that a protein or polypeptide produced by aneoplastic cell in vivo could be altered in sequence from that producedby a tumor cell in cell culture or by a transformed cell. Thus, MNproteins and/or polypeptides which have varying amino acid sequencesincluding without limitation, amino acid substitutions, extensions,deletions, truncations and combinations thereof, fall within the scopeof this invention. It can also be appreciated that a protein extantwithin body fluids is subject to degradative processes, such as,proteolytic processes; thus, MN proteins that are significantlytruncated and MN polypeptides may be found in body fluids, such as,sera. The phrase “MN antigen” is used herein to encompass MN proteinsand/or polypeptides.

It will further be appreciated that the amino acid sequence of MNproteins and polypeptides can be modified by genetic techniques. One ormore amino acids can be deleted or substituted. Such amino acid changesmay not cause any measurable change in the biological activity of theprotein or polypeptide and result in proteins or polypeptides which arewithin the scope of this invention, as well as, MN muteins.

The MN proteins and polypeptides of this invention can be prepared in avariety of ways according to this invention, for example, recombinantly,synthetically or otherwise biologically, that is, by cleaving longerproteins and polypeptides enzymatically and/or chemically. A preferredmethod to prepare MN proteins is by a recombinant means. Particularlypreferred methods of recombinantly producing MN proteins are describedbelow for the GST-MN, MN 20-19, MN-Fc and MN-PA proteins.

Recombinant Production of MN Proteins and Polypeptides

A representative method to prepare the MN proteins shown in FIG. 1 orfragments thereof would be to insert the full-length or an appropriatefragment of MN cDNA into an appropriate expression vector as exemplifiedbelow. In Zavada et al., WO 93/18152, supra, production of a fusionprotein GEX-3X-MN (now termed GST-MN) using the partial cDNA clone(described above) in the vector pGEX-3X (Pharmacia) is described.Nonglycosylated GST-MN (the MN fusion protein MN glutathioneS-transferase) from XL1-Blue cells. Herein described is the recombinantproduction of both a glycosylated MN protein expressed from insect cellsand a nonglycosylated MN protein expressed from E. coli using theexpression plasmid pEt-22b [Novagen Inc.; Madison, Wis. (USA)].

Baculovirus Expression Systems. Recombinant baculovirus express vectorshave been developed for infection into several types of insect cells.For example, recombinant baculoviruses have been developed for amongothers: Aedes aegypti, Autographa californica, Bombyx mor, Drosphilamelanogaster, Heliothis zea, Spodoptera frugiperda, and Trichoplusia ni[PCT Pub. No. WO 89/046699; Wright, Nature, 321: 718 (1986); Fraser etal., In Vitro Cell Dev. Biol., 25: 225 (1989). Methods of introducingexogenous DNA into insect hosts are well-known in the art. DNAtransfection and viral infection procedures usually vary with the insectgenus to be transformed. See, for example, Autographa [Carstens et al.,Virology, 101: 311 (1980)]; Spodoptera [Kang, “Baculovirus Vectors forExpression of Foreign Genes,” in: Advances in Virus Research, 35(1988)]; and Heliothis (virescens) [PCT Pub. No. WO 88/02030].

A wide variety of other host-cloning vector combinations may be usefullyemployed in cloning the MN DNA isolated as described herein. Forexample, useful cloning vehicles may include chromosomal, nonchromosomaland synthetic DNA sequences such as various known bacterial plasmidssuch as pBR322, other E. coli plasmids and their derivatives and widerhost range plasmids such as RP4, phage DNA, such as, the numerousderivatives of phage lambda, e.g., NB989 and vectors derived fromcombinations of plasmids and phage DNAs such as plasmids which have beenmodified to employ phage DNA expression control sequences.

Useful hosts may be eukaryotic or prokaryotic and include bacterialhosts such as E. coli and other bacterial strains, yeasts and otherfungi, animal or plant hosts such as animal or plant cells in culture,insect cells and other hosts. Of course, not all hosts may be equallyefficient. The particular selection of host-cloning vehicle combinationmay be made by those of skill in the art after due consideration of theprinciples set forth herein without departing from the scope of thisinvention.

The following are representative examples of genetically engineering MNproteins of this invention. The descriptions are exemplary and not meantto limit the invention in any way.

Expression of MN 20-19 Protein

A representative, recombinantly produced MN protein of this invention isthe MN 20-19 protein which, when produced in baculovirus-infected Sf9cells [Spodoptera frugiperda cells; Clontech; Palo Alto, Calif. (USA)],is glycosylated. The MN 20-19 protein misses the putative signal peptide(aas 1-37) of SEQ ID NO: 6 (FIG. 1), has a methionine (Met) at theN-terminus for expression, and a Leu-Glu-His-His-His-His-His-His [SEQ.ID NO.: 22] added to the C-terminus for purification.

In order to insert the portion of the MN coding sequence for the GST-MNfusion protein into alternate expression systems, a set of primers forPCR was designed. The primers were constructed to provide restrictionsites at each end of the coding sequence, as well as in-frame start andstop codons. The sequences of the primers, indicating restriction enzymecleavage sites and expression landmarks, are shown below. Primer#20:N-terminus                      

Translation start 5′GTCGCTAGCTCCATGGGTCATATGCAGAGGTTGCCCCGGATGCAG 3′[SEQ. ID. NO. 17]       NheI NcoI    NdeI  

MN cDNA#1 Primer #19:C-terminus            

Translation stop 5′GAAGATCTCTTACTCGAGCATTCTCCAAGATCCAGCCTCTAGG 3′ [SEQ.ID. NO. 18]    BgIII     XhoI   

MN cDNAThe SEQ ID NOS: 17 and 18 primers were used to amplify the MN codingsequence present in the GEX-3X-MN vector using standard PCR techniques.The resulting PCR product (termed MN 20-19) was electrophoresed on a0.5% agarose/1X TBE gel; the 1.3 kb band was excised; and the DNArecovered using the Gene Clean II kit according to the manufacturer'sinstructions [Bio101; LaJolla, Calif. (USA)].

MN 20-19 and plasmid pET-22b were cleaved with the restriction enzymesNdeI and XhoI, phenol-chloroform extracted, and the appropriate bandsrecovered by agarose gel electrophoresis as above. The isolatedfragments were ethanol co-precipitated at a vector:insert ratio of 1:4.After resuspension, the fragments were ligated using T4 DNA ligase. Theresulting product was used to transform competent Novablue E. coli cells[Novagen, Inc.]. Plasmid mini-preps [Magic Minipreps; Promega] from theresultant ampicillin resistant colonies were screened for the presenceof the correct insert by restriction mapping. Insertion of the genefragment into the pET-22b plasmid using the NdeI and XhoI sites added a6-histidine tail to the protein that could be used for affinityisolation.

To prepare MN 20-19 for insertion into the baculovirus expressionsystem, the MN 20-19 gene fragment was excised from pET-22b using therestriction endonucleases XbaI and PvuI. The baculovirus shuttle vectorpBacPAK8 [Clontech] was cleaved with XbaI and PacI. The desiredfragments (1.3 kb for MN 20-19 and 5.5 kb for pBacPAK8) were isolated byagarose gel electrophoresis, recovered using Gene Clean II, andco-precipitated at an insert:vector ratio of 2.4:1.

After ligation with T4 DNA ligase, the DNA was used to transformcompetent NM522 E. coli cells (Stratagene). Plasmid mini-preps fromresultant ampicillin resistant colonies were screened for the presenceof the correct insert by restriction mapping. Plasmid DNA from anappropriate colony and linearized BacPAK6 baculovirus DNA [Clontech]were used to transform Sf9 cells by standard techniques. Recombinationproduced BacPAK viruses carrying the MN 20-19 sequence. Those viruseswere plated onto Sf9 cells and overlaid with agar.

Plaques were picked and plated onto Sf9 cells. The conditioned media andcells were collected. A small aliquot of the conditioned media was setaside for testing. The cells were extracted with PBS with 1% TritonX100.

The conditioned media and the cell extracts were dot blotted ontonitrocellulose paper. The blot was blocked with 5% non-fat dried milk inPBS. Mab M75 were used to detect the MN 20-19 protein in the dot blots.A rabbit anti-mouse Ig-HRP was used to detect bound Mab M75. The blotswere developed with TMB/H₂0₂ with a membrane enhancer [KPL;Gaithersburg, Md. (USA)]. Two clones producing the strongest reaction onthe dot blots were selected for expansion. One was used to produce MN20-19 protein in High Five™ cells [Invitrogen Corp., San Diego, Calif.(USA); BTI-TN-5BI-4; derived from Trichoplusia ni egg cell homogenate].MN 20-19 protein was purified from the conditioned media from the virusinfected High Five™ cells.

The MN 20-19 protein was purified from the conditioned media byimmunoaffinity chromatography. 6.5 mg of Mab M75 was coupled to 1 g ofTresyl activated TOYOPEARL® [solid support in bead form; Tosoh, Japan(#14471)]. Approximately 150 ml of the conditioned media was run throughthe M75-TOYOPEARL® column. The column was washed with PBS, and the MN20-19 protein was eluted with 1.5 M MgCl. The eluted protein was thendialyzed against PBS.

Synthetic and Biologic Production of MN Proteins and Polypeptides

MN proteins and polypeptides of this invention may be prepared not onlyby recombinant means but also by synthetic and by other biologic means.Synthetic formation of the polypeptide or protein requires chemicallysynthesizing the desired chain of amino acids by methods well known inthe art. Exemplary of other biologic means to prepare the desiredpolypeptide or protein is to subject to selective proteolysis a longerMN polypeptide or protein containing the desired amino acid sequence;for example, the longer polypeptide or protein can be split withchemical reagents or with enzymes.

Chemical synthesis of a peptide is conventional in the art and can beaccomplished, for example, by the Merrifield solid phase synthesistechnique [Merrifield, J., Am. Chem. Soc., 85: 2149-2154 (1963); Kent etal., Synthetic Peptides in Biology and Medicine, 29 f.f. eds. Alitalo etal., (Elsevier Science Publishers 1985); and Haug, J. D., AmericanBiotechnology Laboratory, 5(1): 40-47 (January/February 1987)].

Techniques of chemical peptide synthesis include using automatic peptidesynthesizers employing commercially available protected amino acids, forexample, Biosearch [San Rafael, Calif. (USA)] Models 9500 and 9600;Applied Biosystems, Inc. [Foster City, Calif. (USA)] Model 430; Milligen[a division of Millipore Corp.; Bedford, Mass. (USA)] Model 9050; and DuPont's RAMP™ (Rapid Automated Multiple Peptide Synthesis) [Du PontCompass, Wilmington, Del. (USA)].

Identification of MN Protein Partner(s)

A search for protein(s) interacting with MN was initiated usingexpression cloning of the corresponding cDNA(s) and a MN-Fc fusionprotein as a probe. The chimerical MN-Fc cDNA was constructed in pSG5Cvector by substitution of MN cDNA sequences encoding both thetransmembrane anchor and the intracellular tail of MN protein with thecDNA encoding Fc fragment of the mouse IgG. The Fc fragment cDNA wasprepared by RT-PCR from the mouse hybridoma producing IgG2a antibody.

The chimerical MN-Fc cDNA was expressed by transient transfection in COScells. COS cells were transfected using leptofection. Recombinant MN-Fcprotein was released to TC medium of the transfected cells (due to thelack of the transmembrane region), purified by affinity chromatographyon a Protein A Sepharose and used for further experiments.

Protein extracts from mock-transfected cells and the cells transfectedwith pSG5C-MN-Fc were analysed by immunoblotting using the M75 MAb,SwαM-Px and ECL detection® [ECL®—enhanced chemoluminescent system todetect phosphorylated tyrosine residues; Amersham; Arlington, Hts., IL(USA)]. The size of MN-Fc protein expressed from the pSG5C vectorcorresponds to its computer predicted molecular weight.

³⁵S-labeled MN-Fc protein was employed in cell surface binding assay. Itwas found to bind to several mammalian cells, e.g., HeLa, Raji, COS,QT35, BL3. Similar results were obtained in cell adhesion assay usingMN-Fc protein dropped on bacterial Petri dishes. These assays revealedthat KATO III human stomach adenocarcinoma cell line is lacking anability to interact with MN-Fc protein. This finding allowed us to useKATO III cells for expression cloning and screening of the cDNA codingfor MN-binding protein.

The cDNA expression library in pBK-CMV vector was prepared from denseHeLa cells and used for transfection of KATO III cells. For the firstround of screening, KATO III cells were transfected by electroporation.After two days of incubation, the ligand-expressing cells were allowedto bind to MN-Fc protein, then to Protein A conjugated with biotin andfinally selected by pulling down with streptavidin-coated magneticbeads. Plasmid DNA was extracted from the selected cells and transformedto E. coli. Individual E. coli colonies were picked and pools of 8-10clones were prepared. Plasmid DNA from the pools was isolated and usedin the second round of screening.

In the second round of screening, KATO III cells were transfected byDEAE dextran method. To identify the pool containing the cDNA forMN-binding protein, an ELISA method based on the binding of MN-Fc to thetransfected cells, and detection using peroxidase labelled Protein Awere used. Pools are selected by ability to bind MN-Fc.

In the third round of screening, plasmid DNAs isolated from individualbacterial colonies of selected pools are transfected to KATO III cells.The transfected cells are subjected to binding with MN-Fc and detectionwith Protein A as before. Such exemplary screening is expected toidentify a clone containing the cDNA which codes for the putative MNprotein partner. That clone would then be sequenced and the expressionproduct confirmed as binding to MN protein by cell adhesion assay.(Far-Western blotting, co-precipitation etc.) Hybridomas producing Mabsto the expression product would then be prepared which would allow theanalysis of the biological characteristics of the protein partner of MN.

Nucleic Acid Probes and Test Kits

Nucleic acid probes of this invention are those comprising sequencesthat are complementary or substantially complementary to the MN cDNAsequence shown in FIG. 1 or to other MN gene sequences, such as, thecomplete genomic sequence of FIG. 2A-F [SEQ ID NO: 5] and the putativepromoter sequence [SEQ ID NO: 27 of FIG. 6]. The phrase “substantiallycomplementary” is defined herein to have the meaning as it is wellunderstood in the art and, thus, used in the context of standardhybridization conditions. The stringency of hybridization conditions canbe adjusted to control the precision of complementarity. Two nucleicacids are, for example, substantially complementary to each other, ifthey hybridize to each other under stringent hybridization conditions.

Stringent hybridization conditions are considered herein to conform tostandard hybridization conditions understood in the art to be stringent.For example, it is generally understood that stringent conditionsencompass relatively low salt and/or high temperature conditions, suchas provided by 0.02 M to 0.15 M NaCl at temperatures of 50° C. to 70° C.Less stringent conditions, such as, 0.15 M to 0.9 M salt at temperaturesranging from 20° C. to 55° C. can be made more stringent by addingincreasing amounts of formamide, which serves to destabilize hybridduplexes as does increased temperature.

Exemplary stringent hybridization conditions are described in Sambrooket al., Molecular Cloning: A Laboratory Manual, pages 1.91 and 9.47-9.51(Second Edition, Cold Spring Harbor Laboratory Press; Cold SpringHarbor, N.Y.; 1989); Maniatis et al., Molecular Cloning: A LaboratoryManual, pages 387-389 (Cold Spring Harbor Laboratory; Cold SpringHarbor, N.Y.; 1982); Tsuchiya et al., Oral Surgery, Oral Medicine, OralPathology, 71(6): 721-725 (June 1991).

Preferred nucleic acid probes of this invention are fragments of theisolated nucleic acid sequences that encode MN proteins or polypeptidesaccording to this invention. Preferably those probes are composed of atleast 25 nts, more preferably at least 27 nts, still more preferably atleast 29 nts, further preferably at least 50 nts.

Nucleic acid probes of this invention need not hybridize to a codingregion of MN. For example, nucleic acid probes of this invention mayhybridize partially or wholly to a non-coding region of the genomicsequence shown in FIG. 2A-F [SEQ ID NO: 5]. Conventional technology canbe used to determine whether fragments of SEQ ID NO: 5 or relatednucleic acids are useful to identify MN nucleic acid sequences. [See,for example, Benton and Davis, supra and Fuscoe et al., supra.]

Areas of homology of the MN nt sequence to other non-MN nt sequences areindicated above. In general, nucleotide sequences that are not in theAlu or LTR-like regions, of preferably 25 bases or more, or still morepreferably of 50 bases or more, can be routinely tested and screened andfound to hybridize under stringent conditions to only MN nucleotidesequences. Further, not all homologies within the Alu-like MN genomicsequences are so close to Alu repeats as to give a hybridization signalunder stringent hybridization conditions. The percent of homologybetween MN Alu-like regions and a standard Alu-J sequence are indicatedas follows: Region of Homology within MN Genomic Sequence SEQ. [SEQ IDNO: 5; ID. FIG. 2A-F] NOS. % Homology to Entire Alu-J Sequence  921-121259 89.1% 2370-2631 60 78.6% 4587-4880 61 90.1% 6463-6738 62 85.4%7651-7939 63 91.0% 9020-9317 64 69.8% % Homology to One Half of Alu-JSequence 8301-8405 65 88.8% 10040-10122 66  73.2%.

Nucleic acid probes of this invention can be used to detect MN DNAand/or RNA, and thus can be used to test for the presence or absence ofMN genes, and amplification(s), mutation(s) or genetic rearrangements ofMN genes in the cells of a patient. For example, overexpression of an MNgene may be detected by Northern blotting and RNase protection analysisusing probes of this invention. Gene alterations, as amplifications,translocations, inversions, and deletions among others, can be detectedby using probes of this invention for in situ hybridization tochromosomes from a patient's cells, whether in metaphase spreads orinterphase nuclei. Southern blotting could also be used with the probesof this invention to detect amplifications or deletions of MN genes.Restriction Fragment Length Polymorphism (RFLP) analysis using saidprobes is a preferred method of detecting gene alterations, mutationsand deletions. Said probes can also be used to identify MN proteinsand/or polypeptides as well as homologs or near homologs thereto bytheir hybridization to various mRNAs transcribed from MN genes indifferent tissues.

Probes of this invention thus can be usefuldiagnostically/prognostically. Said probes can be embodied in test kits,preferably with appropriate means to enable said probes when hybridizedto an appropriate MN gene or MN mRNA target to be visualized. Suchsamples include tissue specimens including smears, body fluids andtissue and cell extracts.

PCR Assays

RT-PCR is a preferred PCR assay to detect abnormal MN gene expression.For example, mRNA can be detected using PCR primers from the MN cDNA.[Eg., McKiernan et al., Cancer Res., 57: 2362 (1997).]

To detect relatively large genetic rearrangements, hybridization testscan be used. To detect relatively small genetic rearrangements, as, forexample, small deletions or amplifications, or point mutations, PCRwould preferably be used. [U.S. Pat. Nos. 4,800,159; 4,683,195;4,683,202; and Chapter 14 of Sambrook et al., Molecular Cloning: ALaboratory Manual, supra]

An exemplary assay would use cellular DNA from normal and cancerouscells, which DNA would be isolated and amplified employing appropriatePCR primers. The PCR products would be compared, preferably initially,on a sizing gel to detect size changes indicative of certain geneticrearrangements. If no differences in sizes are noted, furthercomparisons can be made, preferably using, for example,PCR-single-strand conformation polymorphism (PCR-SSCP) assay or adenaturing gradient gel electrophoretic assay. [See, for example,Hayashi, K. in PCR Methods and Applications, 1: 34-38 (1991); and Meyerset al., Methods in Enzymology, 155: 501 (1987).]

Assays

Assays according to this invention are provided to detect and/orquantitate MN antigen or MN-specific antibodies in vertebrate samples,preferably mammalian samples, more preferably human samples. Suchsamples include tissue specimens, body fluids, tissue extracts, cells,cell lysates and cell extracts, among other samples. MN antigen may bedetected by immunoassay, immunohistochemical staining, immunoelectronand scanning microscopy using immunogold among other techniques.

Preferred tissue specimens to assay by immunohistochemical staininginclude cell smears, histological sections from biopsied tissues ororgans, and imprint preparations among other tissue samples. Such tissuespecimens can be variously maintained, for example, they can be fresh,frozen, or formalin-, alcohol- or acetone- or otherwise fixed and/orparaffin-embedded and deparaffinized. Biopsied tissue samples can be,for example, those samples removed by aspiration, bite, brush, cone,chorionic villus, endoscopic, excisional, incisional, needle,percutaneous punch, and surface biopsies, among other biopsy techniques.

Preferred cervical tissue specimens include cervical smears, conizationspecimens, histologic sections from hysterectomy specimens or otherbiopsied cervical tissue samples. Preferred means of obtaining cervicalsmears include routine swab, scraping or cytobrush techniques, amongother means. More preferred are cytobrush or swab techniques.Preferably, cell smears are made on microscope slides, fixed, forexample, with 55% EtOH or an alcohol based spray fixative and air-dried.

Papanicolaou-stained cervical smears (Pap smears) can be screened by themethods of this invention, for example, for retrospective studies.Preferably, Pap smears would be decolorized and re-stained with labeledantibodies against MN antigen. Also archival specimens, for example,matched smears and biopsy and/or tumor specimens, can be used forretrospective studies. Prospective studies can also be done with matchedspecimens from patients that have a higher than normal risk ofexhibiting abnormal cervical cytopathology.

Preferred samples in which to assay MN antigen by, for example, Westernblotting or radioimmunoassay, are tissue and/or cell extracts. However,MN antigen, particularly in a soluble form, as the extracellular domain,can be detected in body fluids, which can include among other fluids:blood, serum, plasma, semen, breast exudate, gastric secretions, fecalsuspensions, bile, saliva, tears, sputum, mucous, urine, lymph,cytosols, ascites, pleural effusions, amniotic fluid, bladder washes,bronchioalveolar lavages and cerebrospinal fluid. It is preferred thatthe MN antigen be concentrated from a larger volume of body fluid beforetesting. Preferred body fluids to assay would depend on the type ofcancer for which one was testing, but in general preferred body fluidswould be urine, serum, mucous, gastric secretions, bile, breast exudate,pleural effusions and ascites.

Diagnostic nucleic acid can be labelled, directly or indirectly, bymethods known in the art, and can be used in conventional Southern orNorthern hybridization assays. Such assays can be employed inidentifying transformants or for in vitro diagnosis, such as to detectMN mRNA in tissues as a measure of oncogenic activity. The presence ofMN mRNA or precursors thereto for most tissues being indicative ofoncogenic activity, whereas the absence or a reduced level of MN mRNA instomach and gallbladder tissues in comparison to the levels of mRNAfound in the counterpart normal tissues is considered indicative ofoncogenic activity. DNA which encodes MN proteins can be obtained bychemical synthesis, by screening reverse transcripts of mRNA fromplacental or other cells, or by screening genomic libraries fromeukaryotic cells, among other methods.

MN-specific antibodies can be bound by serologically active MNproteins/polypeptides in samples of such body fluids as blood, plasma,serum, lymph, mucous, tears, urine, spinal fluid and saliva; however,such antibodies are found most usually in blood, plasma and serum,preferably in serum. Correlation of the results from the assays todetect and/or quantitate MN antigen and MN-specific antibodies reactivetherewith, provides a preferred profile of the disease condition of apatient.

The assays of this invention are both diagnostic and/or prognostic,i.e., diagnostic/prognostic. The term “diagnostic/prognostic” is hereindefined to encompass the following processes either individually orcumulatively depending upon the clinical context: determining thepresence of disease, determining the nature of a disease, distinguishingone disease from another, forecasting as to the probable outcome of adisease state, determining the prospect as to recovery from a disease asindicated by the nature and symptoms of a case, monitoring the diseasestatus of a patient, monitoring a patient for recurrence of disease,and/or determining the preferred therapeutic regimen for a patient. Thediagnostic/prognostic methods of this invention are useful, for example,for screening populations for the presence of neoplastic orpre-neoplastic disease, determining the risk of developing neoplasticdisease, diagnosing the presence of neoplastic and/or pre-neoplasticdisease, monitoring the disease status of patients with neoplasticdisease, and/or determining the prognosis for the course of neoplasticdisease. For example, it appears that the intensity of theimmunostaining with MN-specific antibodies may correlate with theseverity of dysplasia present in samples tested.

The present invention is useful for screening for the presence of a widevariety of neoplastic diseases as indicated above. The inventionprovides methods and compositions for evaluating the probability of thepresence of malignant or pre-malignant cells, for example, in a group ofcells freshly removed from a host. Such an assay can be used to detecttumors, quantitate their growth, and help in the diagnosis and prognosisof disease. The assays can also be used to detect the presence of cancermetastasis, as well as confirm the absence or removal of all tumortissue following surgery, cancer chemotherapy and/or radiation therapy.It can further be used to monitor cancer chemotherapy and tumorreappearance.

The presence of MN antigen or antibodies can be detected and/orquantitated using a number of well-defined diagnostic assays. Those inthe art can adapt any of the conventional immunoassay formats to detectand/or quantitate MN antigen and/or antibodies.

Many formats for detection of MN antigen and MN-specific antibodies are,of course available. Those can be Western blots, ELISAs, RIAs,competitive EIA or dual antibody sandwich assays, immunohistochemicalstaining, among other assays all commonly used in the diagnosticindustry. In such immunoassays, the interpretation of the results isbased on the assumption that the antibody or antibody combination willnot cross-react with other proteins and protein fragments present in thesample that are unrelated to MN.

Representative of one type of ELISA test for MN antigen is a formatwherein a microtiter plate is coated with antibodies made to MNproteins/polypeptides or antibodies made to whole cells expressing MNproteins, and to this is added a patient sample, for example, a tissueor cell extract. After a period of incubation permitting any antigen tobind to the antibodies, the plate is washed and another set of anti-MNantibodies which are linked to an enzyme is added, incubated to allowreaction to take place, and the plate is then rewashed. Thereafter,enzyme substrate is added to the microtiter plate and incubated for aperiod of time to allow the enzyme to work on the substrate, and theadsorbance of the final preparation is measured. A large change inabsorbance indicates a positive result.

It is also apparent to one skilled in the art of immunoassays that MNproteins and/or polypeptides can be used to detect and/or quantitate thepresence of MN antigen in the body fluids, tissues and/or cells ofpatients. In one such embodiment, a competition immunoassay is used,wherein the MN protein/polypeptide is labeled and a body fluid is addedto compete the binding of the labeled MN protein/polypeptide toantibodies specific to MN protein/polypeptide.

In another embodiment, an immunometric assay may be used wherein alabeled antibody made to a MN protein or polypeptide is used. In such anassay, the amount of labeled antibody which complexes with theantigen-bound antibody is directly proportional to the amount of MNantigen in the sample.

A representative assay to detect MN-specific antibodies is a competitionassay in which labeled MN protein/polypeptide is precipitated byantibodies in a sample, for example, in combination with monoclonalantibodies recognizing MN proteins/polypeptides. One skilled in the artcould adapt any of the conventional immunoassay formats to detect and/orquantitate MN-specific antibodies. Detection of the binding of saidantibodies to said MN protein/polypeptide could be by many ways known tothose in the art, e.g., in humans with the use of anti-human labeledIgG.

An exemplary immunoassay method of this invention to detect and/orquantitate MN antigen in a vertebrate sample comprises the steps of:

a) incubating said vertebrate sample with one or more sets of antibodies(an antibody or antibodies) that bind to MN antigen wherein one set islabeled or otherwise detectable;

b) examining the incubated sample for the presence of immune complexescomprising MN antigen and said antibodies.

Another exemplary immunoassay method according to this invention is thatwherein a competition immunoassay is used to detect and/or quantitate MNantigen in a vertebrate sample and wherein said method comprises thesteps of:

a) incubating a vertebrate sample with one or more sets of MN-specificantibodies and a certain amount of a labeled or otherwise detectable MNprotein/polypeptide wherein said MN protein/polypeptide competes forbinding to said antibodies with MN antigen present in the sample;

b) examining the incubated sample to determine the amount oflabeled/detectable MN protein/polypeptide bound to said antibodies; and

c) determining from the results of the examination in step b) whether MNantigen is present in said sample and/or the amount of MN antigenpresent in said sample.

Once antibodies (including biologically active antibody fragments)having suitable specificity have been prepared, a wide variety ofimmunological assay methods are available for determining the formationof specific antibody-antigen complexes. Numerous competitive andnon-competitive protein binding assays have been described in thescientific and patent literature, and a large number of such assays arecommercially available. Exemplary immunoassays which are suitable fordetecting a serum antigen include those described in U.S. Pat. Nos.3,984,533; 3,996,345; 4,034,074; and 4,098,876.

Antibodies employed in assays may be labeled or unlabeled. Unlabeledantibodies may be employed in agglutination; labeled antibodies may beemployed in a wide variety of assays, employing a wide variety oflabels.

Suitable detection means include the use of labels such asradionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors, freeradicals, particles, dyes and the like. Such labeled reagents may beused in a variety of well known assays, such as radioimmunoassays,enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and thelike. See for example, U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837;and 4,233,402.

Immunoassay Test Kits

The above outlined assays can be embodied in test kits to detect and/orquantitate MN antigen and/or MN-specific antibodies (includingbiologically active antibody fragments). Kits to detect and/orquantitate MN antigen can comprise MN protein(s)/polypeptides(s) and/orMN-specific antibodies, polyclonal and/or monoclonal. Suchdiagnostic/prognostic test kits can comprise one or more sets ofantibodies, polyclonal and/or monoclonal, for a sandwich format whereinantibodies recognize epitopes on the MN antigen, and one set isappropriately labeled or is otherwise detectable.

Test kits for an assay format wherein there is competition between alabeled (or otherwise detectable) MN protein/polypeptide and MN antigenin the sample, for binding to an antibody, can comprise the combinationof the labeled protein/polypeptide and the antibody in amounts whichprovide for optimum sensitivity and accuracy.

Test kits for MN-specific antibodies preferably compriselabeled/detectable MN proteins(s) and/or polypeptides(s), and maycomprise other components as necessary, such as, controls, buffers,diluents and detergents. Such test kits can have other appropriateformats for conventional assays.

A kit for use in an enzyme-immunoassay typically includes anenzyme-labelled reagent and a substrate for the enzyme. The enzyme can,for example, bind either an MN-specific antibody of this invention or toan antibody to such an MN-specific antibody.

Preparation of MN-Specific Antibodies

The term “antibodies” is defined herein to include not only wholeantibodies but also biologically active fragments of antibodies,preferably fragments containing the antigen binding regions. Furtherincluded in the definition of antibodies are bispecific antibodies thatare specific for MN protein and to another tissue-specific antigen.

Antibodies of the invention may be prepared by conventional methodologyand/or by genetic engineering. Chimeric antibodies that are humanized toreduce antigenicity are preferred for in vivo use. Antibody fragmentsmay be genetically engineered, preferably from the variable regions ofthe light and/or heavy chains (V_(H) and V_(L)), including thehypervariable regions, and still more preferably from both the V_(H) andV_(L) regions. For example, the term “antibodies” as used hereincomprehends polyclonal and monoclonal antibodies and biologically activefragments thereof including among other possibilities “univalent”antibodies [Glennie et al., Nature, 295: 712 (1982)]; Fab proteinsincluding Fab′ and F(ab′)₂ fragments whether covalently ornon-covalently aggregated; light or heavy chains alone, preferablyvariable heavy and light chain regions (V_(H) and V_(L) regions), andmore preferably including the hypervariable regions [otherwise known asthe complementarity determining regions (CDRs) of said V_(H) and V_(L)regions]; Fc proteins; “hybrid” antibodies capable of binding more thanone antigen; constant-variable region chimeras; “composite”immunoglobulins with heavy and light chains of different origins;“altered” antibodies with improved specificity and other characteristicsas prepared by standard recombinant techniques and also byoligonucleotide-directed mutagenesis techniques [Dalbadie-McFarland etal., PNAS (USA), 79: 6409 (1982)].

Bispecific Antibodies. Bispecific antibodies can be produced bychemically coupling two antibodies of the desired specificity.Bispecific MAbs can preferably be developed by somatic hybridization of2 hybridomas. Bispecific MAbs for targeting MN protein and anotherantigen can be produced by fusing a hybridoma that produces MN-specificMAbs with a hybridoma producing MAbs specific to another antigen. Forexample, a cell (a quadroma), formed by fusion of a hybridoma producinga MN-specific MAb and a hybridoma producing an anti-cytotoxic cellantibody, will produce hybrid antibody having specificity of the parentantibodies. [See, e.g., Immunol. Rev. (1979); Cold Spring HarborSymposium Quant. Biol., 41: 793 (1977); van Dijk et al., Int. J. Cancer,43: 344-349 (1989).] Thus, a hybridoma producing a MN-specific MAb canbe fused with a hybridoma producing, for example, an anti-T3 antibody toyield a cell line which produces a MN/T3 bispecific antibody which cantarget cytotoxic T cells to MN-expressing tumor cells.

It may be preferred for therapeutic and/or imaging uses that theantibodies be biologically active antibody fragments, preferablygenetically engineered fragments, more preferably genetically engineeredfragments from the V_(H) and/or V_(L) regions, and still more preferablycomprising the hypervariable regions thereof. However, for sometherapeutic uses bispecific antibodies targeting MN protein andcytotoxic cells would be preferred.

There are conventional techniques for making polyclonal and monoclonalantibodies well-known in the immunoassay art. Immunogens to prepareMN-specific antibodies include MN proteins and/or polypeptides,preferably purified, and MX-infected tumor line cells, for example,MX-infected HeLa cells, among other immunogens.

Anti-peptide antibodies are also made by conventional methods in the artas described in European Patent Publication No. 44,710 (published Jan.27, 1982). Briefly, such anti-peptide antibodies are prepared byselecting a peptide from an MN amino acid sequence as from FIG. 1,chemically synthesizing it, conjugating it to an appropriate immunogenicprotein and injecting it into an appropriate animal, usually a rabbit ora mouse; then, either polyclonal or monoclonal antibodies are made, thelatter by a Kohler-Milstein procedure, for example.

Besides conventional hybridoma technology, newer technologies can beused to produce antibodies according to this invention. For example, theuse of the PCR to clone and express antibody V-genes and phage displaytechnology to select antibody genes encoding fragments with bindingactivities has resulted in the isolation of antibody fragments fromrepertoires of PCR amplified V-genes using immunized mice or humans.[Marks et al., BioTechnology, 10: 779 (July 1992) for references; Chianget al., BioTechniques, 7(4): 360 (1989); Ward et al., Nature, 341: 544(Oct. 12, 1989); Marks et al., J. Mol. Biol. 222: 581 (1991); Clacksonet al., Nature, 352: (15 Aug. 1991); and Mullinax et al., PNAS (USA),87: 8095 (Oct. 1990).]

Descriptions of preparing antibodies, which term is herein defined toinclude biologically active antibody fragments, by recombinanttechniques can be found in U.S. Pat. No. 4,816,567 (issued Mar. 28,1989); European Patent Application Publication Number (EP) 338,745(published Oct. 25, 1989); EP 368,684 (published Jun. 16, 1990); EP239,400 (published Sep. 30, 1987); WO 90/14424 (published Nov. 29,1990); WO 90/14430 (published May 16, 1990); Huse et al., Science. 246:1275 (Dec. 8, 1989); Marks et al., BioTechnology, 10: 779 (July 1992);La Sastry et al., PNAS (USA), 86: 5728 (August 1989); Chiang et al.,BioTechniques, 7(40): 360 (1989); Orlandi et al., PNAS (USA), 86: 3833(May 1989); Ward et al. Nature, 341: 544 (Oct. 12, 1989); Marks et al.,J. Mol. Biol., 222: 581 (1991); and Hoogenboom et al., Nucleic AcidsRes., 19(15): 4133 (1991).

Representative Mabs

Monoclonal antibodies for use in the assays of this invention may beobtained by methods well known in the art for example, Galfre andMilstein, “Preparation of Monoclonal Antibodies: Strategies andProcedures,” in Methods in Enzymology: Immunochemical Techniques, 73:1-46 [Langone and Vanatis (eds); Academic Press (1981)]; and in theclassic reference, Milstein and Kohler, Nature, 256: 495-497 (1975).]

Although representative hybridomas of this invention are formed by thefusion of murine cell lines, human/human hybridomas [Olsson et al., PNAS(USA), 77: 5429 (1980)] and human/murine hybridomas [Schlom et al., PNAS(USA), 77: 6841 (1980); Shearman et al. J. Immunol., 146: 928-935(1991); and Gorman et al., PNAS (USA), 88: 4181-4185 (1991)] can also beprepared among other possibilities. Such humanized monoclonal antibodieswould be preferred monoclonal antibodies for therapeutic and imaginguses.

Monoclonal antibodies specific for this invention can be prepared byimmunizing appropriate mammals, preferably rodents, more preferablyrabbits or mice, with an appropriate immunogen, for example,MaTu-infected HeLa cells, MN fusion proteins, or MNproteins/polypeptides attached to a carrier protein if necessary.Exemplary methods of producing antibodies of this invention aredescribed below.

The monoclonal antibodies useful according to this invention to identifyMN proteins/polypeptides can be labeled in any conventional manner, forexample, with enzymes such as horseradish peroxidase (HRP), fluorescentcompounds, or with radioactive isotopes such as, ¹²⁵I, among otherlabels. A preferred label, according to this invention is ¹²⁵I, and apreferred method of labeling the antibodies is by using chloramine-T[Hunter, W. M., “Radioimmunoassay,” In: Handbook of ExperimentalImmunology, pp. 14.1-14.40 (D. W. Weir ed.; Blackwell,Oxford/London/Edinburgh/Melbourne; 1978)].

Representative mabs of this invention include Mabs M75, MN9, MN12 andMN7 described below. Monoclonal antibodies of this invention serve toidentify MN proteins/polypeptides in various laboratory diagnostictests, for example, in tumor cell cultures or in clinical samples.

Mabs Prepared Against HeLa Cells

MAb M75. Monoclonal antibody M75 (MAb M75) is produced by mouselymphocytic hybridoma VU-M75, which was initially deposited in theCollection of Hybridomas at the Institute of Virology, Slovak Academy ofSciences (Bratislava, Slovakia) and was deposited under ATCC DesignationHB 11128 on Sep. 17, 1992 at the American Type Culture Collection(ATCC). The production of hybridoma VU-M75 is described in Zavada etal., WO 93/18152.

Mab M75 recognizes both the nonglycosylated GST-MN fusion protein andnative MN protein as expressed in CGL3 cells equally well. The M75 MAbrecognizes both native and denatured forms of MN protein [Pastorekova etal. (1992), supra].

Epitopes

The affinity of a MAb to peptides containing an epitope depends on thecontext, e.g. on whether the peptide is a short sequence (4-6 aa), orwhether such a short peptide is flanked by longer aa sequences on one orboth sides, or whether in testing for an epitope, the peptides are insolution or immobilized on a surface. Therefore, it would be expected byones of skill in the art that the representative epitopes describedherein for the MN-specific MAbs would vary in the context of the use ofthose MAbs.

Epitope for M75 MAb

The M75 epitope is considered to be present in at least two copieswithin the 6× tandem repeat of 6 amino acids [aa 61-96 (SEQ ID NO: 97)]in the proteglycan domain of the MN protein. Exemplary peptidesrepresenting that epitope depending on the context may include thefollowing peptides from that tandem repeat: EEDLPS (SEQ ID NO: 10; aa62-67); GEEDLP (SEQ ID NO: 98; aa 61-66; aa 79-84; aa 85-90; aa 91-96);EEDL (SEQ ID NO: 99; aa 62-65; aa 80-83; aa 86-89; aa 92-95); EEDLP (SEQID NO. 100; aa 62-66; aa 80-84; aa 86-90; aa 92-96); EDLPSE (SEQ ID NO:101; aa 63-68); EEDLPSE (SEQ ID NO: 102; aa 62-68); and DLPGEE (SEQ IDNO: 103; aa 82-87, aa 88-93).

Three synthetic peptides from the deduced aa sequence for the EC domainof the MN protein shown in FIG. 1 were prepared. Those syntheticpeptides are represented by aa 51-72 (SEQ ID NO: 104), aa 61-85 (SEQ IDNO: 105) and aa 75-98 (SEQ ID NO.: 106). Each of those syntheticpeptides contains the motif EEDLP (SEQ ID NO: 100) and were shown to bereactive with the M75 MAb.

Mabs Prepared Against Fusion Protein GST-MN

Monoclonal antibodies of this invention were also prepared against theMN glutathione S-transferase fusion protein (GST-MN). BALB/C mice wereimmunized intraperitoneally according to standard procedures with theGST-MN fusion proteinin Freund's adjuvant. Spleen cells of the mice werefused with SP/20 myeloma cells [Milstein and Kohler, supra].

Tissue culture media from the hybridomas were screened against CGL3 andCGL1 membrane extracts in an ELISA employing HRP labelled-rabbitanti-mouse. The membrane extracts were coated onto microtiter plates.Selected were antibodies that reacted with the CGL3 membrane extract.Selected hybridomas were cloned twice by limiting dilution.

The mabs prepared by the just described method were characterized byWestern blots of the GST-MN fusion protein, and with membrane extractsfrom the CGL1 and CGL3 cells. Representative of the mabs prepared areMabs MN9, MN12 and MN7.

Mab MN9. Monoclonal antibody MN9 (Mab MN9) reacts to the same epitope asMab M75, as described above. As Mab M75, Mab MN9 recognizes both theGST-MN fusion protein and native MN protein equally well.

Mabs corresponding to Mab MN9 can be prepared reproducibly by screeninga series of mabs prepared against an MN protein/polypeptide, such as,the GST-MN fusion protein, against the peptides representing the epitopefor Mabs M75 and MN9. Alternatively, the Novatope system [Novagen] orcompetition with the deposited Mab M75 could be used to select mabscomparable to Mabs M75 and MN9.

Mab MN12. Monoclonal antibody MN12 (Mab MN12) is produced by the mouselymphocytic hybridoma MN 12.2.2 which was deposited under ATCCDesignation HB 11647 on Jun. 9, 1994 at the American Type CultureCollection (ATCC). Antibodies corresponding to Mab MN12 can also bemade, analogously to the method outlined above for Mab MN9, by screeninga series of antibodies prepared against an MN protein/polypeptide,against the peptide representing the epitope for Mab MN12. That peptideis aa 55-aa 60 of FIG. 1 [SEQ ID NO: 11]. The Novatope system could alsobe used to find antibodies specific for said epitope.

Mab MN7. Monoclonal antibody MN7 (Mab MN7) was selected from mabsprepared against nonglycosylated GST-MN as described above. Itrecognizes the epitope represented by the amino acid sequence from aa127 to aa 147 [SEQ ID NO: 12] of the FIG. 1 MN protein. Analogously tomethods described above for Mabs MN9 and MN12, mabs corresponding to MabMN7 can be prepared by selecting mabs prepared against an MNprotein/polypeptide that are reactive with the peptide having SEQ ID NO:12, or by the stated alternative means.

Epitope Mapping

Epitope mapping was performed by the Novatope® system, a kit for whichis commercially available from Novagen, Inc. [See, for analogousexample, Li et al., Nature, 363: 85-88 (6 May 1993).] In brief, the MNcDNA was cut into overlapping short fragments of approximately 60 basepairs. The fragments were expressed in E. coli, and the E. coli colonieswere transferred onto nitrocellulose paper, lysed and probed with themab of interest. The MN cDNA of clones reactive with the mab of interestwas sequenced, and the epitopes of the mabs were deduced from theoverlapping polypeptides found to be reactive with each mab.

Therapeutic Use of MN-Specific Antibodies

The MN-specific antibodies of this invention, monoclonal and/orpolyclonal, preferably monoclonal, and as outlined above, may be usedtherapeutically in the treatment of neoplastic and/or pre-neoplasticdisease, either alone or in combination with chemotherapeutic drugs ortoxic agents, such as ricin A. Further preferred for therapeutic usewould be biologically active antibody fragments as described herein.Also preferred MN-specific antibodies for such therapeutic uses would behumanized monoclonal antibodies and/or bispecific antibodies.

MN-specific antibodies can be administered in a therapeuticallyeffective amount, preferably dispersed in a physiologically acceptable,nontoxic liquid vehicle, to patients afflicted withpreneoplastic/neoplastic disease. The MN-specific antibody can be givenalone or as a carrier of an anti-tumor drug. Among the variousantiproliferative, antineoplastic or cytotoxic agents that may be linkedto the MN-specific antibodies are antimetabolites, such as theantifolate, methotrexate, or the purine or pyrimidine analogsmercaptopurine and fluorouracil. Others include antibiotics, lectinssuch as ricin and abrin, toxins such as the subunit of diphtheria toxin,radionuclides such as ²¹¹Astatine and ¹³¹Iodine, radiosensitizers suchas misanidazole or neutron sensitizers such as boron containingorganics. Such agents may be attached to the antibody by conventionaltechniques such as glutaraldehyde cross-linking.

MN-specific antibodies can be used to target cytoxic cells (e.g. human Tcells, monocytes or NK cells). Cytotoxic cells can be attached toMN-expressing tumor cells through Fc receptors on the cytotoxic cells,which bind the Fc portion of a MN-specific antibody, or via a bridgingantibody of dual specificity, that is, a bispecific antibody specificfor MN protein and for the cytotoxic cell.

The cytotoxic cell can be targeted by allowing the bispecific antibodyto bind the cell. After targeting, the cells can be administered to thepatient. Therapy with targeted cells can be used as an adjunct tosurgical therapy, radiation therapy, or chemotherapy.

Anti-Idiotype MN-Specific Antibodies as Tumor Vaccines andAnti-Anti-Idiotype Antibody Sera as Immunotherapeutic

MN-specific anti-idiotype antibodies have therapeutic utility as avaccine for neoplastic disease associated with abnormal MN expression.MN-specific anti-anti-idiotype sera also have therapeutic anti-tumorefficacy. Those therapeutic utilities are demonstrated by research donewith the MN-specific G250 MAb, and anti-idiotype antibodies thereto(Ab2), and further anti-anti-idiotype sera (Ab3) as demonstrated by thestudies described below.

Uemura et al., Biotherapy (Japan) 10(3): 241-244 (1996) (Englishsummary) define an anti-idiotype antibody (Ab2) as “an antibody directedagainst an antigenic determinant located within a variable region of theimmunoglobulin molecule. Ab2 mimicking the normal antigen (so-calledinternal image Ab2) may be used as a surrogate antigen for vaccinationto trigger the host's immune system specifically against the nominalantigen.”

Uemura et al., id., having previously isolated six internal image murineAb2s directed against the G250 MAb—NUH31, 51, 71, 82 (IgG1) and NUH44(IgG2a), explores the application of monoclonal Ab2 as tumor vaccines.Uemura et al. investigated in view of “previous results that RCCtumor-associated-antigen-related idiotype vaccination inducedantigen-specific humoral as well as cellular responses, the antitumorefficacy of anti-anti-idiotype antibody (Ab3) sera obtained from miceimmunized with different internal image Ab2 that . . . mimic theRCC-associated antigen . . . G250 [MN] . . . Nu/nu BALB/c mice carryingsmall established NU12 human RCC xenografts (G250+, 20 mm³) rr receivingan s.c. injection of 2×10⁵ SK-RC-52 (G250+) RCC cells were treated byi.p. injection of 0.2 ml Ab3 sera. This treatment resulted in completetumor rejection and significant tumor growth inhibition as compared tocontrol groups (p<0.01).” Uemura et al. concluded that “immunizationwith Ab2s elicits powerful anti-tumor effects in immunocompetentanimals.”

Uemura et al., J. Urol., 159(5)(Suppl.): Abstract 724 (May 1998),describe MN as an immunotherapeutic target for renal cell carcinoma(RCC). The therapeutic potential of the MN-specific MAb G250 wasevaluated in combination with IFN/IL-2/MCSF (interferon, interleukin-2,macrophage colony stimulating factor) and Ab2 (NUH82)-induced mouseserum (Ab3-82). Ab2s are monoclonal anti-idiotype antibodies raisedagainst MAbG250 which have been shown to be useful as tumor vaccines forRCC.

Uemura et al., id. reported that mice with NUR-2 RCC xenografts weretreated by peri-tumor injection of MAbG250 and/or cytokines or 0.2 ml ofAb3 sera with/without MCSF. The tumor volume in MAbG250 treated animalswas significantly lower than in the controls. IFN or IL-2 treatments wassimilarly effective, but MCSF resulted in no significant tumorinhibition. The IFN/IL-2/MAbG250 therapy increased significantly theanti-tumor effects as compared to MAbG250 or cytokine monotherapy.Further, Ab3-based (Ab2-induced) immunotherapy resulted in tremendoustumor monotherapy growth inhibition as compared to MAbG250 or the othercytokine combination therapies.

MN-Specific Intrabodies—Targeted Tumor Killing Via IntracellularExpression of MN-Specific Antibodies to Block Transport of MN Protein toCell Surface

The gene encoding antibodies can be manipulated so that theantigen-binding domain can be expressed intracellularly. Such“intrabodies” that are targeted to the lumen of the endoplasmicreticulum provide a simple and effective mechanism for inhibiting thetransport of plasma membrane proteins to the cell surface. [Marasco, W.A., “Review—Intrabodies: turning the humoral immune system outside in orintracellular immunization,” Gene Therapy, 4: 11-15 (1997); Chen et al.,“Intracellular antibodies as a new class of therapeutic molecules forgene therapy,” Hum. Gene Ther., 5(5): 595-601 (1994); Mhashilkar et al.,EMBO J., 14: 1542-1551 (1995); Mhashilkar et al., J. Virol., 71:6486-6494 (1997); Marasco (Ed.), Intrabodies: Basic Research andClinical Gene Therapy Applications, (Springer Life Sciences 1998; ISBN3-540-64151-3) (summarizes preclinical studies from laboratoriesworldwide that have used intrabodies); Zanetti and Capra (Eds.),“Intrabodies: From Antibody Genes to Intracellular Communication,” TheAntibodies: Volume 4, [Harwood Academic Publishers; ISBN 90-5702-559-0(Dec. 1997)]; Jones and Marasco, Advanced Drug Delivery Reviews, 31(1-2): 153-170 (1998); Pumphrey and Marasco, Biodrugs, 9(3): 179-185(1998); Dachs et al., Oncology Res., 9(6-7); 313-325 (1997); Rondon andMarasco, Ann. Rev. Microbiol., 51: 257-283 (1997)]; Marasco, W. A.,Immunotechnology, 1(1): 1-19 (1995); and Richardson and Marasco, Trendsin Biotechnology, 13(8): 306-310 (1995).]

MN-specific intrabodies may prevent the maturation and transport of MNprotein to the cell surface and thereby prevent the MN protein fromfunctioning in an oncogenic process. Antibodies directed to MN's EC, TMor IC domains may be useful in this regard. MN protein is considered tomediate signal transduction by transferring signals from the EC domainto the IC tail and then by associating with other intracellular proteinswithin the cell's interior. MN-specific intrabodies could disrupt thatassociation and perturb that MN function.

Inactivating the function of the MN protein could result in reversion oftumor cells to a non-transformed phenotype. [Marasco et al. (1997),supra.] Antisense expression of MN cDNA in cervical carcinoma cells, asdemonstrated herein, has shown that loss of MN protein has led to growthsuppression of the transfected cells. It is similarly expected thatinhibition of MN protein transport to the cell surface would havesimilar effects. Cloning and intracellular expression of the M75 MAb'svariable region is to be studied to confirm that expectation.

Preferably, the intracellularly produced MN-specific antibodies aresingle-chain antibodies, specifically single-chain variable regionfragments or scFv, in which the heavy- and light-chain variable domainsare synthesized as a single polypeptide and are separated by a flexiblelinker peptide, preferably (Gly₄-Ser)₃ [SEQ ID NO: 116].

MN-specific intracellularly produced antibodies can be usedtherapeutically to treat preneoplastic/neoplastic disease bytransfecting preneoplastic/neoplastic cells that are abnormallyexpressing MN protein with a vector comprising a nucleic acid encodingMN-specific antibody variable region fragments, operatively linked to anexpression control sequence. Preferably said expression control sequencewould comprise the MN gene promoter.

Antibody-Mediated Gene Transfer Using MN-Specific Antibodies or Peptidesfor Targeting MN-Expressing Tumor Cells

An MN-specific antibody or peptide covalently linked to polylysine, apolycation able to compact DNA and neutralize its negative charges,would be expected to deliver efficiently biologically active DNA into anMN-expressing tumor cell. If the packed DNA contains the HSVtk geneunder control of the MN promoter, the system would have doublespecificity for recognition and expression only in MN-expressing tumorcells. The packed DNA could also code for cytokines to induce CTLactivity, or for other biologically active molecules.

The M75 MAb (or, for example, as a single chain antibody, or as itsvariable region) is exemplary of such a MN-specific antibody. Example 5discloses heptapeptides (SEQ ID NOS: 107-109) that bind to the enzymaticcenter of the CA domain of the MN protein and, selected peptides orproteins comprising such heptapeptides would also be expected to bind toa binding site on the extracellular domain of the MN protein.

Imaging Use of Antibodies

Further, the MN-specific antibodies of this invention when linked to animaging agent, such as a radionuclide, can be used for imaging.Biologically active antibody fragments or humanized monoclonalantibodies, may be preferred for imaging use.

A patient's neoplastic tissue can be identified as, for example, sitesof transformed stem cells, of tumors and locations of any metastases.Antibodies, appropriately labeled or linked to an imaging agent, can beinjected in a physiologically acceptable carrier into a patient, and thebinding of the antibodies can be detected by a method appropriate to thelabel or imaging agent, for example, by scintigraphy. Exemplary arestudies with the G250 Mab.

Steffens et al., J. Urol., 159(5)(Suppl.): Abstract 562 (May 1998),describe a Phase I/II study with ¹³¹I-cG250 MAb in patients withmetastasized RCC. MAb cG250 is a chimeric MAb in which constant regionsof the mouse immunoglobulin have been exchanged for human immunoglobulinregions. [Oosterwijk and Debruyne, World J. Urol., 13: 186 (1995).]Uptake of the cG250 MAb in primary RCC was shown to be as high as 0.52percent of the injected dose per gram of tumor tissue (% ID/g). Thestudy concluded that “¹³¹I-cG250 is a promising candidate forradioimmunotherapy and a phase I/II activity dose escalation study wasinitiated to determine the safety, maximum tolerable dose (MTD) andtherapeutic potential of ¹³¹I-cG250.”

Bander et al., Proceedings Am. Urol. Assoc., 155(Suppl.): 583A (Abstract1088) (May 1996), describes renal cancer imaging with the MN-specificMAb G250, which detects MN present in 85-90% of renal cancers but doesnot detect MN on normal kidney cells. Bander et al. reports that 48patients were entered in clinical trials with ¹³¹I-G250 MAb.

Antisense MN Nucleic Acid Sequences

MN genes are herein considered putative oncogenes and the encodedproteins thereby are considered to be putative oncoproteins. Antisensenucleic acid sequences complementary to mRNA transcribed from MN genes,as represented by the antisense oligodeoxynucleotides ODN1 and ODN2 [SEQID NOS: 3 and 4] can be used to reduce or prevent expression of the MNgene. [Zamecnick, P. C., pp. 1-6, Prospects for Antisense Nucleic AcidTherapy of Cancer and AIDS, (Wiley-Liss, Inc., New York, N.Y., USA;1991); Wickstrom, E., pp. 7-24, id.; Leserman et al., pp. 25-34, id.;Yokoyama, K., pp. 35-52, id.; van den Berg et al., pp. 63-70, id.;Mercola, D., pp. 83-114, id.; Inouye, Gene, 72: 25-34 (1988); Miller andTs'o, Ann. Reports Med. Chem., 23: 295-304 (1988); Stein and Cohen,Cancer Res., 48: 2659-2668 (1988); Stevenson and Inversen, J. Gen.Virol., 70: 2673-2682 (1989); Goodchild, pp. 53-77,Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression (Cohen,J. S., ed; CRC Press, Boca Raton, Fla., USA; 1989); Dervan et al., pp.197-210, id.; Neckers, L. M., pp. 211-232, id.; Leitner et al., PNAS(USA), 87: 3430-3434 (1990); Bevilacqua et al., PNAS (USA), 85: 831-835(1988); Loke et al. Curr. Top. Microbiol. Immunol., 141: 282-288 (1988);Sarin et al., PNAS (USA), 85: 7448-7451 (1988); Agrawal et al.,“Antisense Oligonucleotides: A Possible Approach for Chemotherapy andAIDS,” International Union of Biochemistry Conference on Nucleic AcidTherapeutics (Jan. 13-17,1991; Clearwater Beach, Fla., USA); Armstrong,L., Ber. Week, pp. 88-89 (Mar. 5, 1990); and Weintraub et al., Trends,1: 22-25 (1985).]

Antisense nucleic acid sequences, preferably oligonucleotides, byhybridizing preferably under stringent hybridization conditions, to MNmRNA, particularly at the 5′ end of the mRNA, corresponding preferablyto the transcription initiation site (i.e., the first 5′ nt of mRNA withlinked terminal 7-methylquanylate), inhibits translation of the mRNA. Amajor transcription initiation site was determined by RNase protectionassay at position 42 upstream from the translation initiation site (nt3465 of genomic sequence). Several minor transcription initiation siteshave also been located.

Particularly preferred are MN antisense nucleic acids, particularlyODNs, that are complementary to the 5′ leader sequence of mRNA (i.e.,nts positioned between cap and translation initiation site).Transcription initiation sites can be detected by standard methods bysearching for the 5′ end of mRNA by RNase protection assay, primerextension, and S1 nuclease mapping (or RACE). The 5′ end of mRNA can bededuced from the full-length cDNA.

The use of MN antisense nucleic acid sequences may be considered to be aform of cancer therapy. Example 11 of Zavada et al., WO 93/18152(published 16 Sep. 1993) describes an in vitro screening procedure topredict if an MN antisense nucleic acid would have therapeutic utilityin vivo. An MN antisense nucleic acid is added to the media of anMN-expressing cell line. After a period of incubation, extracts from thecells are analysed by immunoblotting with labeled MAb M75. Inhibition ofMN protein synthesis relative to control cells is considered predictiveof in vivo therapeutic efficacy.

The experiments with sense/antisense MN cDNA promoter constructsreported above also reasonably predict in vivo therapeutic efficacy ofMN antisense nucleic acids. Transfection with an antisense MN cDNAconstruct has been shown to lower the proliferation rate of anMN-expressing, tumorigenic cell line CGL3 and reduce colony size.Analogous experiments with similar results were obtained by transfectionof SiHa and HeLa cells.

The 29-mer ODN1 and 19-mer ODN2 [SEQ ID NOS: 3 and 4] are representativeof the many antisense nucleic acid sequences that can function toinhibit MN gene expression. Ones of ordinary skill in the art coulddetermine appropriate antisense nucleic acid sequences, preferablyantisense oligonucleotides, from the nucleic acid sequences of FIGS. 1and 2.

Vaccines

It will be readily appreciated that MN proteins and polypeptides of thisinvention can be incorporated into vaccines capable of inducingprotective immunity against neoplastic disease and a dampening effectupon tumorigenic activity. Efficacy of a representative MN fusionprotein GST-MN as a vaccine in a rat model is shown in Example 2.

MN proteins and/or polypeptides may be synthesized or preparedrecombinantly or otherwise biologically, to comprise one or more aminoacid sequences corresponding to one or more epitopes of the MN proteinseither in monomeric or multimeric form. Those proteins and/orpolypeptides may then be incorporated into vaccines capable of inducingprotective immunity. Techniques for enhancing the antigenicity of suchpolypeptides include incorporation into a multimeric structure, bindingto a highly immunogenic protein carrier, for example, keyhole limpethemocyanin (KLH), or diphtheria toxoid, and administration incombination with adjuvants or any other enhancers of immune response.

Preferred MN proteins/polypeptides to be used in a vaccine according tothis invention would be genetically engineered MN proteins. Preferredrecombinant MN protein are the GST-MN, MN 20-19, MN-Fc and MN-PAproteins.

Other exemplary vaccines include vaccinia-MN (live vaccinia virus withfull-length MN cDNA), and baculovirus-MN (full length MN cDNA insertedinto baculovirus vector, e.g. in suspension of infected insect cells).Different vaccines may be combined and vaccination periods can beprolonged.

A preferred exemplary use of such a vaccine of this invention would beits administration to patients whose MN-carrying primary cancer had beensurgically removed. The vaccine may induce active immunity in thepatients and prevent recidivism or metastasis.

It will further be appreciated that anti-idiotype antibodies toantibodies to MN proteins/polypeptides are also useful as vaccines andcan be similarly formulated. As noted above, anti-idiotype antibodies toMN-specific antibodies mimic MN protein/polypeptide. Compositionscomprising such anti-idiotype antibodies may be preferred vaccines ofthis invention when formulated in a physiologically acceptableformulation.

An amino acid sequence corresponding to an epitope of an MNprotein/polypeptide either in monomeric or multimeric form may also beobtained by chemical synthetic means or by purification from biologicalsources including genetically modified microorganisms or their culturemedia. [See Lerner, Sci. Am. 248(2): 66-74 (1983).] Theprotein/polypeptide may be combined in an amino acid sequence with otherproteins/polypeptides including fragments of other proteins, as forexample, when synthesized as a fusion protein, or linked to otherantigenic or non-antigenic polypeptides of synthetic or biologicalorigin. In some instances, it may be desirable to fuse a MN protein orpolypeptide to an immunogenic and/or antigenic protein or polypeptide,for example, to stimulate efficacy of a MN-based vaccine.

The term “corresponding to an epitope of an MN protein/polypeptide” willbe understood to include the practical possibility that, in someinstances, amino acid sequence variations of a naturally occurringprotein or polypeptide may be antigenic and confer protective immunityagainst neoplastic disease and/or anti-tumorigenic effects. Possiblesequence variations include, without limitation, amino acidsubstitutions, extensions, deletions, truncations, interpolations andcombinations thereof. Such variations fall within the contemplated scopeof the invention provided the protein or polypeptide containing them isimmunogenic and antibodies elicited by such a polypeptide or proteincross-react with naturally occurring MN proteins and polypeptides to asufficient extent to provide protective immunity and/or anti-tumorigenicactivity when administered as a vaccine.

Such vaccine compositions will be combined with a physiologicallyacceptable medium, including immunologically acceptable diluents andcarriers as well as commonly employed adjuvants such as Freund'sComplete Adjuvant, saponin, alum, and the like. Administration would bein immunologically effective amounts of the MN proteins or polypeptides,preferably in quantities providing unit doses of from 0.01 to 10.0micrograms of immunologically active MN protein and/or polypeptide perkilogram of the recipient's body weight. Total protective doses mayrange from 0.1 to about 100 micrograms of antigen. Routes ofadministration, antigen dose, number and frequency of injections are allmatters of optimization within the scope of the ordinary skill in theart.

The following examples are for purposes of illustration only and notmeant to limit the invention in any way.

EXAMPLE 1 Immunohistochemical Staining of Tissue Specimens

To study and evaluate the tissue distribution range and expression of MNproteins, the monoclonal antibody M75 was used to stainimmunohistochemically a variety of human tissue specimens. The primaryantibody used in these immunohistochemical staining experiments was theM75 monoclonal antibody. A biotinylated second antibody andstreptavidin-peroxidase were used to detect the M75 reactivity insections of formalin-fixed, paraffin-embedded tissue samples. Acommercially available amplification kit, specifically the DAKO LSAB™kit [DAKO Corp., Carpinteria, Calif. (USA)] which provides matched,ready made blocking reagent, secondary antibody andsteptavidin-horseradish peroxidase was used in these experiments.

M75 immunoreactivity was tested according to the methods of thisinvention in multiple-tissue sections of breast, colon, cervical, lungand normal tissues. Such multiple-tissue sections were cut from paraffinblocks of tissues called “sausages” that were purchased from the City ofHope [Duarte, Calif. (USA)]. Combined in such a multiple-tissue sectionwere normal, benign and malignant specimens of a given tissue; forexample, about a score of tissue samples of breast cancers fromdifferent patients, a similar number of benign breast tissue samples,and normal breast tissue samples would be combined in one suchmultiple-breast-tissue section. The normal multiple-tissue sectionscontained only normal tissues from various organs, for example, liver,spleen, lung, kidney, adrenal gland, brain, prostate, pancreas, thyroid,ovary, and testis.

Also screened for MN gene expression were multiple individual specimensfrom cervical cancers, bladder cancers, renal cell cancers, and head andneck cancers. Such specimens were obtained from U.C. Davis MedicalCenter in Sacramento, Calif. and from Dr. Shu Y. Liao [Department ofPathology; St. Joseph Hospital; Orange, Calif. (USA)].

Controls used in these experiments were the cell lines CGL3 (H/F-Thybrid cells) and CGL1 (H/F-N hybrid cells) which are known to stainrespectively, positively and negatively with the M75 monoclonalantibody. The M75 monoclonal antibody was diluted to a 1:5000 dilutionwherein the diluent was either PBS [0.05 M phosphate buffered saline(0.15 M NaCl), pH 7.2-7.4] or PBS containing 1% protease-free BSA as aprotein stabilizer.

Immunohistochemical Staining Protocol

The immunohistochemical staining protocol was followed according to themanufacturer's instructions for the DAKO LSAB™ kit. In brief, thesections were dewaxed, rehydrated and blocked to remove non-specificreactivity as well as endogenous peroxidase activity. Each section wasthen incubated with dilutions of the M75 monoclonal antibody. After theunbound M75 was removed by rinsing the section, the section wassequentially reacted with a biotinylated antimouse IgG antibody andstreptavidin conjugated to horseradish peroxidase; a rinsing step wasincluded between those two reactions and after the second reaction.Following the last rinse, the antibody-enzyme complexes were detected byreaction with an insoluble chromogen (diaminobenzidine) and hydrogenperoxide. A positive result was indicated by the formation of aninsoluble reddish-brown precipitate at the site of the primary antibodyreaction. The sections were then rinsed, counterstained withhematoxylin, dehydrated and cover slipped. Then the sections wereexamined using standard light microscopy.

Interpretation. A deposit of a reddish brown precipitate over the plasmamembrane was taken as evidence that the M75 antibody had bound to a MNantigen in the tissue. The known positive control (CGL3) had to bestained to validate the assay. Section thickness was taken intoconsideration to compare staining intensities, as thicker sectionsproduce greater staining intensity independently of other assayparameters.

Results

Preliminary examination of cervical specimens showed that 62 of 68squamous cell carcinoma specimens (91.2%) stained positively with M75.Additionally, 2 of 6 adenocarcinomas and 2 of 2 adenosquamous cancers ofthe cervix also stained positively. In early studies, 55.6% (10 of 18)of cervical dysplasias stained positively. A total of 9 specimensincluding both cervical dysplasias and tumors, exhibited some MNexpression in normal appearing areas of the endocervical glandularepithelium, usually at the basal layer. In some specimens, whereasmorphologically normal-looking areas showed expression of MN antigen,areas exhibiting dysplasia and/or malignancy did not show MN expression.

M75 positive immunoreactivity was most often localized to the plasmamembrane of cells, with the most apparent stain being present at thejunctions between adjacent cells. Cytoplasmic staining was also evidentin some cells; however, plasma membrane staining was most often used asthe main criterion of positivity.

M75 positive cells tended to be near areas showing keratindifferentiation in cervical specimens. In some specimens, positivestaining cells were located in the center of nests of non-stainingcells. Often, there was very little, if any, obvious morphologicaldifference between staining cells and non-staining cells. In somespecimens, the positive staining cells were associated with adjacentareas of necrosis.

In most of the squamous cell carcinomas of the cervix, the M75immunoreactivity was focal in distribution, i.e., only certain areas ofthe specimen stained. Although the distribution of positive reactivitywithin a given specimen was rather sporadic, the intensity of thereactivity was usually very strong. In most of the adenocarcinomas ofthe cervix, the staining pattern was more homogeneous, with the majorityof the specimen staining positively.

Among the normal tissue samples, intense, positive and specific M75immunoreactivity was observed only in normal stomach tissues, withdiminishing reactivity in the small intestine, appendix and colon. Noother normal tissue stained extensively positively for M75.Occasionally, however, foci of intensely staining cells were observed innormal intestine samples (usually at the base of the crypts) or weresometimes seen in morphologically normal appearing areas of theepithelium of cervical specimens exhibiting dysplasia and/or malignancy.In such, normal appearing areas of cervical specimens, positive stainingwas seen in focal areas of the basal layer of the ectocervicalepithelium or in the basal layer of endocervical glandular epithelium.In one normal specimen of human skin, cytoplasmic MN staining wasobserved in the basal layer. The basal layers of these epithelia areusually areas of proliferation, suggesting the MN expression may beinvolved in cellular growth. In a few cervical biopsied specimens, MNpositivity was observed in the morphologically normal appearingstratified squamous epithelium, sometimes associated with cellsundergoing koilocytic changes.

Some colon adenomas (4 of 11) and adenocarcinomas (9 of 15) werepositively stained. One normal colon specimen was positive at the baseof the crypts. Of 15 colon cancer specimens, 4 adenocarcinomas and 5metastatic lesions were MN positive. Fewer malignant breast cancers (3of 25) and ovarian cancer specimens (3 of 15) were positively stained.Of 4 head and neck cancers, 3 stained very intensely with M75.

Although normal stomach tissue was routinely positive, 4 adenocarcinomasof the stomach were MN negative. Of 3 bladder cancer specimens (1adenocarcinoma, 1 non-papillary transitional cell carcinoma, and 1squamous cell carcinoma), only the squamous cell carcinoma was MNpositive. Approximately 40% (12 of 30) of lung cancer specimens werepositive; 2 of 4 undifferentiated carcinomas; 3 of 8 adenocarcinomas; 2of 8 oat cell carcinomas; and, 5 of 10 squamous cell carcinomas. Onehundred percent (4 of 4) of the renal cell carcinomas were MN positive.

In summary, MN antigen, as detected by M75 and immunohistochemistry inthe experiments described above, was shown to be prevalent in tumorcells, most notably in tissues of cervical cancers. MN antigen was alsofound in some cells of normal tissues, and sometimes in morphologicallynormal appearing areas of specimens exhibiting dysplasia and/ormalignancy. However, MN is not usually extensively expressed in mostnormal tissues, except for stomach tissues where it is extensivelyexpressed and in the tissues of the lower gastrointestinal tract whereit is less extensively expressed. MN expression is most often localizedto the cellular plasma membrane of tumor cells and may play a role inintercellular communication or cell adhesion. Representative results ofexperiments performed as described above are tabulated in Table 2. TABLE2 Immunoreactivity of M75 in Various Tissues POS/NEG TISSUE TYPE(#pos/#tested) liver, spleen, lung, normal NEG (all) kidney, adrenalgland, brain, prostate, pancreas, thyroid, ovary, testis skin normal POS(in basal layer) (1/1) stomach normal POS small intestine normal POScolon normal POS breast normal NEG (0/10) cervix normal NEG (0/2) breastbenign NEG (0/17) colon benign POS (4/11) cervix benign POS (10/18)breast malignant POS (3/25) colon malignant POS (9/15) ovarian malignantPOS (3/15) lung malignant POS (12/30) bladder malignant POS (1/3) head &neck malignant POS (3/4) kidney malignant POS (4/4) stomach malignantNEG (0/4) cervix malignant POS (62/68)

The results recorded in this example indicate that the presence of MNproteins in a tissue sample from a patient may, in general, dependingupon the tissue involved, be a marker signaling that a pre-neoplastic orneoplastic process is occurring. Thus, one may conclude from theseresults that diagnostic/prognostic methods that detect MN antigen may beparticularly useful for screening patient samples for a number ofcancers which can thereby be detected at a pre-neoplastic stage or at anearly stage prior to obvious morphologic changes associated withdysplasia and/or malignancy being evident or being evident on awidespread basis.

EXAMPLE 2 Vaccine—Rat Model

As shown in Example 7 of WO 93/18152 (International Publication Date: 16Sep. 1993), in some rat tumors, for example, the XC tumor cell line(cells from a rat rhabdomyosarcoma), a rat MN protein, related to humanMN, is expressed. Thus a model was afforded to study antitumor immunityinduced by experimental MN-based vaccines. The following representativeexperiments were performed.

Nine- to eleven-day-old Wistar rats from several families wererandomized, injected intraperitoneally with 0.1 ml of either control ratsera (the C group) or with rat serum against the MN fusion proteinGST-MN (the IM group). Simultaneously both groups were injectedsubcutaneously with 10⁶ XC tumor cells.

Four weeks later, the rats were sacrificed, and their tumors weighed.The results indicated that the difference between the two groups—C andIM—was significant by Mann-Whitney rank test (U=84, α<0.025). The IMgroup of baby rats developed tumors about one-half the size of thecontrols, and 5 of the 18 passively immunized rats developed no tumor atall, compared to 1 of 18 controls.

EXAMPLE 3 Transient Transformation of Mammalian Cells by MN Protein

This example (1) examines the biological consequences of transfectinghuman or mouse cells with MN-cDNA inserted into expression vectors,mainly from the viewpoint of the involvement of MN protein inoncogenesis; (2) determines if MN protein exerts carbonic anhydraseactivity, and whether such activity is relevant for morphologictransformation of cells; and (3) tests whether MN protein is a celladhesion molecule (CAM).

Synopsis

Methods: MN-cDNA was inserted into 3 expression vectors and was used fortransfecting human or mouse cells. MN protein was detected by Westernblotting, radioimmunoassay or immunoperoxidase staining; in all teststhe MN-specific monoclonal antibody M75 (MAb M75) was used. Carbonicanhydrase activity was determined by the acidification velocity ofcarbonate buffer in CO₂ atmosphere.

Results: (1) Cells (human CGL-1 and mouse NIH3T3 cells) transfected withMN-cDNA showed morphologic transformation, but reverted to normalphenotype after 4-5 weeks. (2) This reversion was not due to the loss,silencing or mutation of the MN insert. (3) MN protein has the enzymeactivity of a carbonic anhydrase, which can be inhibited withacetazolamide; however, the inhibition of the carbonic anhydrase enzymeactivity did not affect transformation. (4) MN protein is an adhesionprotein, involved in cell-to-cell contacts.

Background

This example concerns transformation of mammalian cells by MN-cDNAinserted into expression vectors derived from retroviruses. Such vectorsare suitable for efficient and stable integration into cellular DNA andfor continuous expression of MN protein. Cells transfected with theseconstructs showed morphologic transformation, but after some time, theyreverted to normal phenotype.

Sulfonamides, including acetazolamide, are very potent inhibitors ofknown carbonic anhydrases [Maren and Ellison, Mol. Pharmacol., 3:503-508 (1967)]. Acetazolamide was tested to determine if it inhibitedalso the MN-carbonic anhydrase, and if so, whether inhibition of theenzyme affected cell transformation.

There are reasons to believe that MN protein could be involved in directcell-to-cell interactions: A) previous observations indicated afunctional resemblance of MN protein to surface glycoproteins ofenveloped viruses, which mediate virus adsorption to cell surfacereceptors, and MN participated in the formation of phenotypically mixedvirions of vesicular stomatitis virus. B) Inducibility of MN proteinexpression by growing HeLa cells in densely packed monolayers suggeststhat it may be involved in direct interactions between cells. C)Finally, there is a structural similarity between the MN protein andreceptor tyrosine phosphatase β, which also contains proteoglycan andcarbonic anhydrase domains; those domains mediate direct contactsbetween cells of the developing nervous system [Peles et al., Cell, 82:251-260 (1995)]. Therefore, MN protein was tested to see if it bound tocell surface receptors; the result was clearly positive that it does.

Materials and Methods

Cell Lines

Cells used in this example were: CGL1 and CGL3-respectivelynon-tumorigenic and tumorigenic HeLa×fibroblast hybrids [Stanbridge etal., Somat. Cell Genet., 7: 699-712 (1981)], mouse cell line NIH3T3,HeLa cells and monkey Vero cells. The NIH3T3 cells were seeded at verylow density to obtain colonies started from single cells. The mostnormal appearing colony, designated subclone 2, was picked for use inthe experiments reported in this example.

Expression Vectors

Full-length MN cDNA was acquired from a pBluescript subclone [Pastoreket al., Oncogene, 9: 2877-2888 (1994)]. To remove 5′ and 3′ noncodingsequences, that might reduce subsequent gene expression, a polymerasechain reaction (PCR) was performed. The 5′ primerTAGACAGATCTACGATGGCTCCCCTGTGCCCCAG [SEQ ID NO: 88] encompasses atranslation start site and Bg1II cloning site, and the 3′ primerATTCCTCTAGACAGTTACCGGCTCCCCCTCAGAT [SEQ ID NO: 89] encompasses a stopcodon and XbaI cloning site. Full-length MN-cDNA as a template and PfuDNA Polymerase [Stratagene; LaJolla, Calif. (USA)] were used in thereaction.

The PCR product was sequenced and found to be identical with thetemplate; it carried no mutations. The PCR product harbouring solely theMN coding sequence was inserted into three vectors: 1. pMAMneo[Clontech; Palo Alto, Calif. (USA)] plasmid allowingdexamethasone-inducible expression driven by the MMTV-Long TerminalRepeat (LTR) promoter and containing a neo gene for selection oftransformants in media supplemented with Geneticin (G418) antibiotics.2. Retroviral expression vector pGD [Daley et al., Science, 247: 824-829(1990); kindly provided by Prof. David Baltimore, New York-Cambridge)]containing MLV-LTR promoter and neo gene for G418 antibiotics selection.3. Vaccinia virus expression vector pSC11 [Chakrabarti et al., Mol.Cell. Biol., 5: 3403-3409 (1985)]. Transfection was performed via acalcium-phosphate precipitate according to Sambrook et al. (eds.),Molecular cloning, A laboratory manual, 2nd ed., Cold Spring HarborLaboratory Press (1989).

Vaccinia virus strain Praha clone 13 was used as parental virus[Kutinova et al., Vaccine, 13: 487-493 (1995)]. Vaccinia virusrecombinant was prepared by a standard procedure [Perkus et al.,Virology, 152: 285-297 (1986)]. Recombinant viruses were selected andplaque purified twice in rat thymidine-kinase-less RAT2 cells [Topp, W.C., Virology, 113: 408411 (1981)] in the presence of5′-bromodeoxyuridine (100 μg/ml). Blue plaques were identified byoverlaying with agar containing5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (200 μg/ml).

CA Assay

Carbonic anhydrase activity was measured by a micro-method [Brion etal., Anal. Biochem., 175: 289-297 (1988)]. In principle, velocity of thereaction CO₂+H₂O→H₂CO₃ is measured by the time required foracidification of carbonate buffer, detected with phenol red as a pHindicator. This reaction proceeds even in absence of the enzyme, witht₀=control time (this was set to 60 seconds). Carbonic anhydrase reducesthe time of acidification to t; one unit of the enzyme activity reducesthe time to one half of control time: t/t₀=½.

For the experiment, MN protein was immunoprecipitated with Mab M75 fromRIPA buffer (1% Triton X-100, 0.1% deoxycholate, 1 mMphenylmethylsulfonyl-fluoride and 200 trypsin-inhibiting units/ml ofTrasylol in PBS, pH 7.2) extract of Vero cells infected with vaccinia-MNconstruct, after the cells developed cytopathic effect, or with “empty”vaccinia as a control. The MN+antibody complex was subsequently adsorbedto protein A—Staphylococcus aureus cells [Kessler, S. W., J. Immunol.,115: 1617-1624 (1975)] and rinsed 2× with PBS and 2× with 1 mM carbonatebuffer, pH 8.0. The precipitate was resuspended in the same buffer andadded to the reaction mixture. Acetazolamide (Sigma) was tested forinhibition of carbonic anhydrase [Maren and Ellison, supra]. In extractsof infected cells used for immunoprecipitation, the concentration oftotal proteins was determined by the Lowry method [Lowry et al., J.Biol. Chem., 193: 265-275 (1951)] and that of MN protein by acompetition radioimmunoassay as described in Zavada et al., Int. J.Cancer, 54: 268-274 (1993).

Western Blots

Western blotting and development of the blots using ¹²⁵I-labelled M75and autoradiography was performed as before [Pastorekova et al.,Virology, 187: 620-626 (1992); and Zavada (1993), supra].

Adhesion Assay

For the adhesion assay [Hoffman S., “Assays of cell adhesion,” IN:Cell-cell Interactions, (Stevenson et al. eds.) pp. 1-30 (IRL Press atOxford University Press; Oxford, N.Y., Tokyo; 1992)], 25 μl aliquots MNprotein (affinity purified pGEX-3X MN) [Zavada et al. (1993), supra] orof control proteins were spotted on 5 cm-diameter bacteriological Petridishes and allowed to bind for 2 hours at room temperature. This yieldedcircular protein-coated areas of 4-5 mm diameter. MN protein was dilutedto 10 μg/ml in 50 mM carbonate buffer, pH 9.2. Patches of adsorbedcontrol proteins were prepared similarly. Those included collagens typeI and IV, fibronectin, laminin and gelatin (Sigma products), diluted andadsorbed according to the manufacturer's recommendations; FCS and BSAwere also included. After aspiration of the drops, the dishes wererinsed 2× with PBS and saturated for 1 hour with DMEM supplied with 5%FCS. The plates were seeded with 5×10⁵ cells in 5 ml of DMEM+5% FCS andincubated overnight at 37° C. The plates were rinsed with PBS, and theattached cells were fixed with formaldehyde, post-fixed with methanoland Giemsa stained.

Results

1. Transformation and Reversion of CGL1 Cells Transfected with MN-cDNA

Since the expression of MN protein correlated with the tumorigenicity ofHeLa×fibroblast hybrids [Zavada et al. (1993), supra], thenon-tumorigenic hybrid CGL1 cells were first tested. Those cells,transfected with the pMAM.MN construct, after selection with Geneticin,formed colonies with varying degrees of transformation; some of themappeared normal. While normal CGL1 cells are contact inhibited, growingin a parallel orientation, the transformed cells formed very densecolonies, showing the loss of contact inhibition. Such colonies grewmore slowly than the original CGL 1.

After subcloning, the cells isolated from transformed coloniessegregated revertants. The reversion was a gradual, step-wise process;there were colonies with different degrees of reversion. After 2passages, all the cell population became morphologicallyindistinguishable from normal CGL1. This was due to the reversion ofsome cells and to the selective advantage of the revertants, which grewfaster than the transformed cells. Despite repeated attempts, not evenone single stably transformed cell clone was obtained. No transformedcolonies were found in CGL1 cells transfected with an “empty” pMAMcontrol plasmid.

Growth of the CGL1+pMAM.MN revertants in media supplied with 5 μg/ml ofdexamethasone for 7 days enhanced the production of MN protein, but themorphology of the cells did not return to transformed.

2. Rescue of Transforming MN from the Revertants

The reversion of MN-transformed cells to normal phenotype could have atleast 4 causes: A) loss of the MN insert; B) silencing of the MN insert,e.g., by methylation; C) mutation of the MN insert; D) activation of asuppressor gene, coding for a product which neutralizes transformingactivity of MN protein; E) loss of a MN-binding protein. To decide amongthose alternatives, the following experiment was designed as graphicallyoutlined in FIG. 9.

MN-cDNA was inserted into pGD, a vector derived from mouse leukemiavirus—MLV. A defective virus was thereby engineered, which contained theMN gene and the selective marker neo instead of genes coding for viralstructural proteins. With this construct, mouse NIH3T3 cells weretransfected. In media supplied with Geneticin, the cells formed colonieswith phenotypes ranging from strongly transformed to apparently normal.All of the transformed colonies and about 50% of the normal coloniesexpressed MN protein. Contrasting with normal NIH3T3 cells, thetransformants were also able to form colonies in soft agar, reflectiveof the loss of anchorage dependence, characteristic of celltransformation. Upon passaging, the cells isolated from transformedcolonies reverted to normal morphology, and at the same time, they lostthe capacity to form colonies in soft agar, while still expressing theMN protein. This permanent presence of MN protein in revertants ruledout alternatives A) and B) supra, that is, loss or silencing of the MNgene as a cause of reversion.

To decide among the other 3 alternatives, the revertants weresuperinfected with live, replication competent MLV. This virus grows inNIH3T3 cells without any morphologic manifestations, and it works as a“helper” for the pGD.MN construct. Virus progeny from MLV-infectedrevertants represents an artificial virus complex [PGD.MN+MLV]. Thisconsists of 2 types of virions: of standard type MLV particles andvirions containing the pGD.MN genome, enveloped in structural proteinsprovided by the “helper” virus. This virus complex was infectious forfresh NIH3T3 cells; it again induced in them morphologic transformationand the capacity to form agar colonies.

Contrasting with NIH3T3 transfected with PGD.MN, all the colonies ofcells infected with [pGD.MN+MLV] complex, which grew in the presence ofGeneticin, were uniformly transformed and contained MN proteins. Thetransformants once more reverted to normal phenotype although they keptproducing infectious [pGD.MN+MLV] complex, which induced transformationin fresh NIH3T3 cells. This cycle of infection-transformation-reversionwas repeated 3 times with the same result. This ruled out alternativeC)—mutation of MN-cDNA as a cause of reversion.

Normal NIH3T3 cells formed a contact inhibited monolayer of flat cells,which did not stain with Mab M75 and immunoperoxidase. Cells infectedwith [PGD.MN+MLV] complex were clearly transformed: they grew in achaotic pattern and showed loss of contact inhibition. Some of the cellsshowed signs of apoptosis. Two passages later, the cell populationtotally reverted to original phenotype as a result of frequent emergenceof revertants and of their selective advantages (faster growth and ahigher efficiency of plating). In fact, the revertants appeared to growto a somewhat lower saturation density than the original NIH3T3 cells,showing a higher degree of contact inhibition.

The control NIH3T3 cells did not contain any MN protein (Western blot);while both transformed cells and revertants contained the same amountand the same proportion of 54 and 58 kDa bands of MN protein. In anon-reducing gel, MN protein was present in the form of oligomers of 153kDa. Consistently, by competition RIA, approximately 40 ng MN/mg totalprotein was found in both of the transformed cells and revertants.

3. Carbonic Anhydrase Activity and its Inhibition

Since the carbonic anhydrase domain represents a considerable part ofthe MN protein (see FIG. 8), tests were performed to determine whetherit is indeed enzymatically active. Vero cells infected with thevaccinia.MN construct, which contained more of the MN protein than othercells used in the present experiments, served as a source of MN protein.The cells were extracted with RIPA buffer, and MN protein wasconcentrated and partially purified by precipitation with MAb M75 andSAC. The immunoprecipitate was tested for CA activity. 78 μl ofprecipitate contained 1 unit of the enzyme. From the extract, theconcentration of total proteins and of MN protein was determined; 1 unitof enzyme corresponded to 145 ng of MN protein or to 0.83 mg of totalprotein. The immunoprecipitate from Vero cells infected with controlvirus had no enzyme activity.

Activity of MN carbonic anhydrase was inhibited by acetazolamide;1.53×10⁸M concentration of the drug reduced enzyme activity to 50%.

Preliminary tests showed that confluent cultures of HeLa or of NIH3T3cells tolerated 10⁻⁵-10⁻³M concentration of acetazolamide for 3 dayswithout any signs of toxicity and without any effect on cell morphology.In sparse cultures, 10⁻⁵M acetazolamide did not inhibit cell growth, but10M already caused a partial inhibition. Thus, 10⁻⁵M acetazolamide wasadded to NIH3T3 cells freshly transformed with the [pGD.MN+MLV] complex.After 4 days of incubation, the colonies were fixed and stained. Nodifference was seen between cells growing in the presence or absence ofacetazolamide; both were indistinguishable from correctly transformedNIH3T3 cells. Thus, the enzymatic activity of carbonic anhydrase is notrelevant for the transforming activity of MN protein.

4. Cell Adhesion Assay

To determine whether or not MN protein is a cell adhesion molecule(CAM), adhesion assays were performed in plastic bacteriological Petridishes (not treated for use with tissue culture). Cells do not adhere tothe surfaces of such dishes, unless the dishes are coated with a bindingprotein. NIH3T3 cells adhered, spread and grew on patches of adsorbed MNprotein. Only very few cells attached outside the areas coated with MNprotein.

Other variants of the experiment demonstrated that NIH3T3 cells adheredand spread on patches of adsorbed collagen I and IV, fibronectin andlaminin. NIH3T3 cells did not attach to dots of adsorbed gelatin, FCS orBSA.

CGL1, HeLa and Vero cells also adhered to MN protein, but 3 leukemiacell lines showed no adherence. CGL3 cells, strongly expressing MNprotein; adhered less efficiently to MN protein dots then did CGL1. Thepresence of 10⁻⁴M acetazolamide in the media did not affect the celladhesion.

To confirm the specificity of adhesion, MN protein was absorbed with SACloaded with MAb M75 (directed to MN) or MAb M67, directed to anunrelated antigen (Pastorekova et al., supra), before it was applied tothe surface of the Petri dishes. Absorption with the SAC-M75 complextotally abrogated the cell binding activity, whereas absorption withSAC-M67 was without any effect.

Additional Cell Adhesion Results

A shortened MN, missing TM and IC segments, is shed into the medium by5ET1 cells (a HeLa×fibroblast hybrid, analogous to CGL3 cells thatexpress MN protein abundantly) or by Vero cells infected with W carryingMN-cDNA with deleted TM and IC sequences. The shed MN protein waspurified from the media, and tested in cell adhesion assays. The cellsadhered, spread and grew only on the patches covered with adsorbedcomplete MN protein, but not on the dots of MN lacking TM and ICregions. Analogous results have been described also for some otheradhesion molecules. A variety of cells (NIH3T3, CGL1, CGL3, HeLa, XC)attached to MN protein dots suggesting that the MN receptor(s) is commonon the surface of vertebrate cells.

Tests were also performed with extracellular matrix proteins or controlproteins dotted on nitrocellulose. The dot-blots were treated with MNprotein solution. Bound MN protein was detected with MAb M75. MN proteinabsorbed to the dots of collagen I and IV, but not to fibronectin,laminin, gelatine or BSA.

Discussion

The data provided herein is consistent with the view that the MN genemost likely represents a novel type of oncogene or proto-oncogene. MNprotein has a very strong association with certain types of humancarcinomas, and is absent from almost all normal tissues (except asshown infra). Cells are morphologically transformed by MN-cDNA, suchtransformation being characterized by increased cell density, acriss-cross pattern of cell growth and the acquisition of the capacityto form colonies in soft agar (Pastorek et al., supra). Those featuresare characteristic of cells transformed with tumor viruses or withcloned oncogenes [Todaro et al., PNAS (USA) 51: 66-73 (1964); Macphersonand Montagnier, Virology, 23: 291-294 (1964)].

Adhesion molecules mediate cell-to-cell or cell to extracellular matrixbinding; they play an essential role in embryogenesis and in cell growthand differentiation. Recent data has demonstrated that besides theirmechanical function, some CAMs are also involved in signal transductioncascades. Their up-regulation or ectopic expression leads to disruptionof the normal program of cell differentiation. CAMs are believed to playa role in invasion and metastasis as well as in the early steps ofcarcinogenesis [Pigott and Power, The Adhesion Molecule (Academic Press,London (1993); Rosales et al., Biochem. Biophys. Acta., 1242: 77-98(1995)]. Therefore, the present finding of MN being an adhesion moleculeappears very plausible. The development of tumors is generally amulti-step process; in cervical carcinomas, papillomaviruses certainlyplay an important rule [Zur Hausen et al., Virology, 184: 9-13 (1991)].The ectopic expression of MN protein could represent an additional step.A third factor participating in the genesis of cervical carcinomas ismost likely the loss or inactivation of tumor suppressor genes.

The extracellular part of MN protein contains domains homologous toproteoglycans and to CAs. Both of those are known to be engaged incell-to-cell contacts, or in binding of cells to the extracellularmatrix. Receptor protein tyrosine phosphatase β is a cell membraneprotein in embryo brains, binding by its CA domain to contactin on thesurface of neurons, and by its proteoglycan domain to the glia [Peles etal., (1995) supra]. Another example is vaccinia virus, containing a CAdomain in its surface glycoprotein, which is responsible for virusattachment to cellular receptors [Maa et al., J. Biol. Chem., 265:1569-1577 (1990)]. Both of those structures related to CAs areenzymatically inactive. Due to mutations in the active center, theycannot bind Zn²⁺ ions, but its pocket-like structure was preserved, withpotential to accommodate other ligands than CO₂+H₂₀.

The CA and proteoglycan domains of the MN protein could, like in theabove mentioned proteins, be involved in cell-to-cell interactions. Itsderegulated expression could upset correct communication among cells.Years ago, contact inhibition was shown to be mediated by directinteractions between cells [Stoker, M. G. P., J. Cell. Sci., 2: 293-304(1967], but its molecular mechanism has not been satisfactorilyelucidated up to now. MN protein may interfere in signal transmissionestablishing contact inhibition.

Reversion of tumor cells to normal phenotype was first described inhamster cells transformed with Rous sarcoma virus. The cells changedtheir chaotic growth back to the original parallel array [Macpherson,I., Science. 148: 1731-1733 (1965)]. The src oncogene wastranscriptionally silenced in segregated revertants. The provirus wasshown to be methylated, and the methylation is known to preventtranscription [Searle et al., Nucl. Acid. Res. 12: 5193-5210 (1984)].Reversion was also found in other tumor cells, which was again due tothe methylation of the src gene [Hejnar et al., Cell Growth Differ., 5:277-284 (1994)]. The frequency of reversion in those cases was of theorder of 10⁻²/cell/division.

Methylation of the MN insert was clearly not the cause of reversion inthe system presented here, since the revertants contained the MNprotein. In some instances, the reversion was explained by mutations ofthe src oncogene [Oppermann et al., Virology, 108: 47-70 (1981)], but inMN, mutation did not account for the reversion in MN-transformed cells.

All the MN-transformed cells revert within 4-5 weeks. A plausible (butnot the only) explanation could be that the MN protein expressed intransfected cells, after some interval, switches on a suppressorgene(s), which code(s) for a hypothetical “normalizing” protein. This,in turn, neutralizes the transforming activity of the MN protein. Thispostulated suppressor gene is switched off in normal cells, notcontaining MN protein, and it is lost or inactivated in tumor cells.

Hybridization of HeLa cells with normal fibroblasts [Stanbridge et al.,supra] indicated that HeLa cells express a critical oncogene,responsible for tumorigenicity in nude mice. This oncogene iscounteracted by a tumor suppressor gene (or genes), which is absent orinactive in HeLa cells, but is functioning in fibroblasts. The originalHeLa×fibroblast hybrid was non-tumorigenic, but it segregated cloneswith restored tumorigenicity. Tumorigenic segregants lost chromosome 11,containing the putative suppressor gene.

The MN protein is a candidate for being the product of the criticaloncogene; its expression in the hybrids has been shown to correlate withtheir tumorigenicity [e.g., Zavada et al. (1993), supra]. The presentresults indicate that additional mechanisms might exist, which are ableto “heal” a cancerous cell. Understanding the molecular mechanisms ofaction of MN protein in normal and in tumor cells and elucidating howthe reversion works may provide new approaches to cancer therapy.

Prospects for therapy. There are many new principles of cancer therapyemploying oncoproteins or molecules that interact with them as targets[Mendelsohn and Lippman, “Principles of molecular cell biology ofcancer: growth factors,” In: DeVita et al., eds., Cancer: principles andpractice of oncology, pp. 114-133 4th ed., Philadelphia: Lippinocott(1993); DeVita et al., eds., Biologic therapy of cancer, 2nd ed.,Philadelphia: Lippinocott (1995)]. The MN protein and at least some ofits ligands (or receptors) appear to be particularly suitable for suchpurposes. MN protein is located on the cell surface and is thusvulnerable. It is present in a high percentage of certain human tumors,but is normally expressed abundantly only in the gastric and gallbladdermucosa as shown infra.

EXAMPLE 4 Identification of MN's Binding Site

MN protein is a tumor-associated cell adhesion molecule (CAM). Toidentify its binding site, a series of overlapping oligopeptides,spanning the N-terminal domain of the MN protein were synthesized. TheN-terminal domain is homologous to that of proteoglycans and contains atandem repeat of six amino acids.

The series of oligopeptides were tested by the cell adhesion assayprocedure essentially as described above in Example 3. The syntheticoligopeptides were immobilized on hydrophobic plastic surfaces to see ifthey would mediate the attachment, spreading and growth of cells. Alsoinvestigated were whether the oligopeptides or antibodies inhibitedattachment of cells (NIH3T3, HeLa and CGL1) to purified MN proteincoated onto such plastic surfaces. The MN protein was affinity purifiedon agarose covalently linked to sulfonamide, as the MN proteinencompasses a CA domain.

Several of the oligopeptides were found to be biologically active: (i)when immobilized onto the plastic, they mediate attachment of cells(NIH3T3, HeLa and to CGL1); (ii) when added to the media, they competefor attachment to cells with the immobilized MN protein; (iii) theseoligopeptides, present in the media do not inhibit attachment of cellsto TC plastic, but they prevent cell-cell adhesion and formation ofintercellular contacts; (iv) treatment of immobilized MN protein and ofactive peptides with MAb M75 abrogates their affinity for the cells; and(v) the binding site of MN was determined to be closely related oridentical to the epitope for MAb M75, at least two copies of which arelocated in the 6-fold tandem repeat of 6 amino acids [aa 61-96 (SEQ IDNO: 97)] in the proteoglycan domain of MN protein.

It was concluded that ectopically expressed MN protein most likelyparticipates in oncogenesis by intervention into normal cell-cellcontacts. MN's binding site represents a potential target for whichtherapeutic agents can be designed.

EXAMPLE 5 Identification of Peptides Binding to MN Protein Using PhageDisplay

(a) To identify peptides that are recognized by MN protein, aheptapeptide phage display library [Ph.D.®-7 Peptide 7-mer Library Kit(phage display peptide library kit); New England Biolabs; Beverly, Mass.(USA)] was screened. In screening the library, a selection process,i.e., biopanning [Parmley and Smith, Gene, 73: 308 (1988); Noren, C. J.,NEB Transcript, 8(1): 1 (1996)] was carried out by incubating the phagesencoding the peptides with a plate coated with MN protein, washing awaythe unbound phage, eluting and amplifying the specifically bound phage.

The target MN protein in this process was a glutathione-S-transferase(GST) MN fusion protein (GST-MN). GST-MN is a recombinantly producedfusion protein expressed from pGEX-3X-MN containing the cDNA for the MNprotein without the signal peptide. GST-MN was produced in bacteriaunder modified cultivation conditions (decreased optical density,decreased temperature). Such cultivation prevented premature terminationof translation and resulted in synthesis of the protein molecules whichwere in vast majority of the full length. The GST-MN protein was usedfor coating of the wells and binding the relevant phages. The boundphages were then eluted by acetazolamide, amplified and used for twoadditional rounds of screening.

After sequencing of several independent phage clones obtained after thethird round of screening, the following heptapeptides were obtained: (1)GETRAPL (SEQ ID NO: 107) (2) GETREPL (SEQ ID NO: 108) (3) GQTRSPL (SEQID NO: 109) (4) GQTRSPL (SEQ ID NO: 109) (5) GQTRSPL (SEQ ID NO: 109)(6) GQTRSPL (SEQ ID NO: 109) (7) GQTRSPL (SEQ ID NO: 109)The heptapeptides show very similar or identical sequences indicatingthat the binding is specific. The fact that phages bearing theseheptapeptides were eluted by acetazolamide, an inhibitor of carbonicanhydrase activity, indicates that the peptides bind to the CA domain ofMN protein.

(b) Analogous screening of the heptapeptide phage display library isdone using collagen 1, shown to bind MN protein, for elution of phages.Different peptide(s) binding to different part(s) of the MN proteinmolecule are expected to be identified. After identifying suchMN-binding peptides, the corresponding synthetic peptides shall then beanalysed for their biological effects.

EXAMPLE 6 Analysis of Stomach cDNA Sequence and Expression in Tissues ofHuman and Rat Alimentary Tracts

Pastorekova et al., Gastroenterology, 112: 398408 (1997), provides ananalysis of the stomach cDNA sequence and its expression in tissues ofthe human and rat alimentary tracts. The aim of the study was todetermine if there were differences in cDNAs, to obtain an overview ofdistribution in the alimentary tract and to obtain data on expression intumors.

Methods. A MN cDNA isolated from a human stomach library was sequencedalong with the cDNA derived from HeLa cells. Western blotting andimmunohistochemical analyses of human and animal tissues were performedusing MN-specific M75 MAb and rabbit antiserum to human CA II.

Results. Sequence analysis showed no differences between the stomach-and HeLa-derived cDNAs. MN was detected at the basolateral surface ofgastric, intestinal, and gallbladder epithelia. In stomach tumorsamples, expression of MN was lost or reduced.

Conclusions. Differential distribution of MN in normal and tumor tissuesis not associated with cDNA mutations. Evolutionary conservation invertebrates as well as abundant expression of MN protein in normal humangastric mucosa, but not in derived tumors, indicate its physiologicalimportance.

Details of the Materials and Methods used and a detailed discussion ofthe results of this study can be found in Pastorekova et al., id.Summarized below in Table 3 is the immunostaining data on MN'sdistribution in the human and rat alimentary tracts. TABLE 3 Summary ofthe Distribution of MN/CA IX in the Human and Rat Alimentary TractOrgans Historical site Rat Human Oral mucosa surface epithelial cellsn.d. − Parotid gland serous cells − − duct cells − − Submandibularserous cells − − gland mucous cells − − duct cells − − Esophagus surfaceepithelial cells n.d. − mucous glands n.d. − Stomach surface epithelialcells +++ +++ parietal cells +++ +++ zymogen cells +++ +++ Duodenumsurface epithelial cells ++ n.d. Brunner's glands − n.d. Jejunum surfacenongoblet epithelial cells − n.d. goblet cells − n.d. Ileum surfacenongoblet epithelial cells − + goblet cells − + Colon (proximal) surfacenongoblet epithelial cells +++ + goblet cells +++ + Colon (middle)surface nongoblet epithelial cells ++ + goblet cells ++ + Colon (distal)surface nongoblet epithelial cells + n.d. goblet cells + n.d. Liverhepatocytes − − duct cells + ++ Gallbladder luminal epithelial cells n.d+++ Pancreas zymogen cells − − islets of Langerhans − − duct cells − +−, no staining;+, weak staining;++, moderate staining;+++, intense staining;n.d., not done.

Data was obtained by immunohistochemical staining of several tissuesamples. The intestinal metaplasia of the gastric mucosa showed a faintpositive staining for MN/CA IX which is, however, considerably weakerthan that of normal stomach mucosa. No positive reaction for MN/CA IXwas seen in a sample from a gastric adenocarcinoma, while the neoplasticepithelial cells of the same sample showed intense signal for human CAII. Four additional samples of stomach carcinomas were found to benegative for MN/CA IX. These data indicates that the expression of MN/CAIX is lost or considerably reduced in stomach tumor cells.

EXAMPLE 7 MN/CA IX Expression in Colorectal Tumors

In the normal human colon, MN/CA IX is expressed in the proliferatingzone of the crypt epithelium with gradual decrease in the distalcolorectum. Saarnio et al., Am. J. Pathol., 153(1): 279 (1998), reportson a study of MN expression in a series of colorectal neoplasms and acomparative analysis of MN and Ki-67 expression in colorectal tumors.Ki-67, a nuclear protein, has been reported as a reliable marker of cellproliferation in the gastrointestinal mucosa. [Lee et al., Cancer, 78:1881-1897 (1996); Holt et al., Cancer Epidemiol, Biomarkers Prev., 6:131-135 (1997).] The comparative analysis confirmed that MN is expressedin areas with high proliferative capacity, and indicates that MN hasutility as a marker for a cell proliferation in the colorectal mucosa.The increase and abnormal localization of MN expression observed incolorectal tumors points to MN's functional involvement in thepathogenesis of colorectal tumors.

Specimens

Studied were 69 colonic lesions from 60 patients, including 8hyperplastic polyps, 1 juvenile polyp, 39 adenomas, and 21adenocarcinomas. Seven metastases of colorectal adenocarcinomas werealso analyzed, including six mesenteric lymph node metastases and oneliver metastasis. The adenomatous lesions included 18 tubular, 11tubulovillous, and 4 villous tumors. The grade of dysplasia was low in13 lesions, moderate in 19, and high in 8. Some tumors showed varyingdysplasia or adenomatous and invasive histology in the same lesion.There were three patients with familial adenomatous polyposis diseasefrom whom two samples were analyzed, one from the cecum and the otherfrom the rectum.

The group of 21 malignant colorectal tumors consisted of 6 welldifferentiated, 9 moderately differentiated, and 6 poorly differentiatedadenocarcinomas. There were 6 adenocarcinomas with a mucinous component.There were 2 carcinomas of stage A in Dukes' classification, 10 at stageB, 7 at stage C, and 2 at stage D. The primary lesions had been isolatedfrom the right colon (n=13), transverse colon (n=19), descending colon(n=8), sigmoid colon (n=13), and rectum (n=16).

Antibodies and Immunostaining

The MN-specific MAb M75 was used. Polyclonal anti-CA VI serum was usedas a control since CA VI has the greatest homology to MN, but is notexpressed in the gut. Additional controls were obtained by omitting thefirst antibody from immunostaining. Sections were immunostainedaccording to the biotin-streptavidin complex method as described above.The intensity of staining for MN was scored by two of the investigatorson a scale of 0 to 2 as follows: 0, absent or weak focal reaction; 1,weak reaction; 2 moderate to strong reaction. The distribution ofimmunoreactivity between the superficial and deep parts of the mucosawas recorded separately.

To assess proliferative activity, the serial sections were stained witha primary antibody to Ki-67 (clone MIB-1, PharMingen, San Diego, Calif.)and detected by a biotin-streptavidin-peroxidase system, and thesections were counterstained with Harris' hematoxylin. The number ofMIB-1 positive cells was counted in both the superficial and the deephalf of the mucosa. All positively stained nuclei, regardless ofintensity, were regarded as positive. An approximation to the Ki-67score (labeling index) was obtained for each sample as the percentage oftumor cells counted that had positively stained nuclei.

Sixteen samples containing areas of normal mucosa were used to calculatethe control labeling index for Ki-67. The mean value of the score forthe proliferative zone of the normal mucosa was 37% with a 95%confidence interval from 28% to 46%. To allow comparison withplasma-membrane-associated MN, the immunoreactivities of the nuclearKi-67 were adjusted to the same scale of 0 to 2, on which 0 representsless than 28% (weak staining), 1 represents 28% to 46% (moderatestaining), and 2 represents more than 46% (strong staining). Furtherdetails on the Materials and Methods used in this study can be found inSaarnio et al., Am. J. Pathol., 153(1): 279-285 (1998). TABLE 4 Summaryof the Immunohistochemical Staining for MN in the Normal ColorectalMucosa, Colorectal Tumors, and Metastases of Colorectal Carcinomas MeanMN Mean MN Mean SF (median, DE (median, MN n range) range) SF/DE Normalepithelium 16   0 (0, 0-0) 0.3 (0, 0-1) 0 Hyperplastic 8 0.1 (0, 0-1)1.1 (1, 0-2) 0.1 polyps Adenomatous 39 1.1 (1, 0-2) 0.5 (0, 0-2) 2.3lesions SD 13 0.9 (1, 0-2) 0.2 (0, 0-2) 4.5 MD 19 1.3 (1, 0-2)  0.6 (10,0-2) 2.2 GD 8 1.0 (1, 0-2) 0.7 (1, 0-2) 1.7 Malignant lesions 21 1.2 (2,0-2) 1.3 (2, 0-2) 1.0 Grade I 6 1.7 (2, 1-2) 1.5 (2, 1-2) 1.0 Grade II 91.1 (2, 0-2) 1.2 (2, 0-2) 0.9 Grade III 6 0.8 (1, 0-2) 0.8 (1, 0-2) 1.0Metastases 7 0.6 (0, 0-2) 0.6 (0, 0-2) 1.0SF, superficial epithelial staining;DE, deep epithelial staining;SD, slight dysplasia;MD, moderate dysplasia;GD, grave dysplasia.

TABLE 5 Summary of the Immunohistochemical Staining for Ki-67 in theNormal Colorectal Mucosa, Colorectal Tumors, and Metasases of ColorectalCarcinomas Mean Mean Ki-67 SF Mean Ki-67 DE Ki-67 n (median, range)(median, range) SF/DE Normal epithelium 16   0 (0, 0-0) 0.9 (1, 0-2) 0Hyperplastic 8   0 (0, 0-0) 2.0 (2, 2-2) 0 polyps Adenomatous 39 1.7 (2,1-2) 0.5 (0, 0-2) 3.4 lesions SD 13 1.7 (2, 1-2) 0.2 (0, 0-1) 8.5 MD 191.6 (2, 1-2) 0.5 (0, 0-2) 3.2 GD 8 1.9 (2, 1-2) 1.1 (1, 5, 0-2) 1.7Malignant lesions 21 1.8 (2, 1-2) 1.5 (2, 0-2) 1.2 Grade I 6 2.0 (2,2-2) 1.7 (2, 1-2) 1.2 Grade II 9 1.9 (1.5, 1-2) 1.7 (1.5, 1-2) 1.1 GradeIII 6 1.4 (1, 0-2) 1.2 (1, 0-2) 1.2 Metastases 7 0.6 (1, 0-2) 0.6 (1,0-2) 1.0SF, superficial epithelial staining;DE, deep epithelial staining;SD, slight dysplasia;MD, moderate dysplasia;GD, grave dysplasia.Results

An outline of the MN and Ki-67 immunoreactivities estimated in thecolorectal lesions is presented in Tables 4 and 5. The intensity of MNimmunostaining was compared with Ki-67 in the superficial and deep partsof the lesions. There was a significant correlation between MN and Ki-67immunoreactivity for superficial (C=0.30, P<0.01) and for cryptal(C=0.31, P<0.01) staining (Spearman).

Normal Mucosa. Sixteen samples contained normal mucosa in which MNstaining was generally quite weak or absent. The staining was localizedto the basolateral plasma membrane of the cryptal epithelial cells, andit was also these cells that showed high Ki-67 immunoreactivity.Occasional epithelial cells in the superficial mucosa expressed Ki-67(mean, 3%), whereas no reaction was detected for MN.

Nonneoplastic Polyps of the Large Intestine. The expression of MN wasstudied in one juvenile polyp and eight hyperplastic polyps. Theimmunostaining of the juvenile polyp for MN was negative. Mosthyperplastic polyps showed a weak or moderate reaction only in the deepparts of the cryptal epithelium, and two of them did not show anyimmunoreactivity at all. There were no differences in staining intensitybetween the proximal and distal polyps. The cryptal cells also showed anintense immunoreaction for Ki-67.

Adenomatous Polyps

The 39 adenomatous lesions were obtained from 30 patients, including 3with familial adenomatous polyposis disease. No staining for MN wasfound in eight lesions (20%), which were located mainly in the distalpart of the colon and rectum. Nineteen lesions (49%) showed weakstaining and twelve (31%) a moderate to strong reaction, located mainlyin the basolateral plasma membrane of the superficial epithelium. TheMN-positive lesions were evenly distributed along with thecranial-caudal axis of the large intestine. No differences in theimmunoreactivity were observed between the adenomas obtained frompatients with familial adenomatous polyposis and sporadic adenomas.

There were 13, 19, and 8 lesions showing low, moderate, or severedysplasia, respectively. In the group with low dysplasia, eight lesionsstained for MN in the superficial part of the mucosa but only two in thecryptal area as well. Furthermore, 17 adenomas with moderate dysplasiawere stained superficially, whereas the cryptal epithelium was alsopositive in 9 lesions. Six of the eight adenomas with severe dysplasiagave a positive signal. The staining for MN was more diffuse in the moredysplastic adenomas, all of which expressed it in the superficialepithelium and five in the cryptal area. Differences in the stainingintensity, however, were not statistically significant (Mann-Whitneytest).

Both the Ki-67 and MN markers show an extension in staining from thecrypts to the surface in adenomatous lesions relative to the normalepithelium.

Malignant Lesions

The material included 21 malignant colorectal tumours, 6 welldifferentiated adenocarcinomas, 9 moderately differentiated, and 6poorly differentiated. The stage according to Dukes' classification wasA in 2 carcinomas, B in 10, C in 7, and D in 2.

No staining for MN was observed in 5 carcinomas (24%), whereas 6 (29%)showed a weak positive reaction and 10 (47%) a moderate to strongreaction. The immunostaining was localized to the plasma membrane of thetumor cells. Interestingly, the most intense signals were seen in fiveof the six adenocarcinomas with a mucinous component. Staining intensitydid not correlate with the location of the carcinoma, but it showed atrend to correlate with the histological grade, being higher in the welldifferentiated tumors. The mean staining intensities were 1.7, 1.2, and0.8 in grades 1, 2, and 3, respectively (grade 1/grade 3, P=0.05,Mann-Whitney). Similarly, the mean staining intensity was higher (1.5)in less advanced tumors (Dukes A plus B, n=12) than in more advancedones (1.0; Dukes C plus D, n=9; P<0.047, Mann-Whitney test). The MNstaining was generally diffuse, being present in both the superficialand deep parts of the mucosa. A diffuse staining pattern was alsocharacteristic of Ki-67. Interestingly, desmoplastic connective tissuein the malignant lesions occasionally showed prominent immunostainingfor MN.

The data on simultaneous expression of Ki-67 and MN in both thesuperficial and deep parts of the mucosa show that the staining for bothantigens is more intense in malignant tumors than in the normal mucosa,with only a few exceptions.

Six mesenteric lymph node metastases of colorectal carcinoma and oneliver metastasis was studied. Three of six lymph node metastases werepositive in the same manner as their primary tumors. The livermetastasis and its primary carcinoma also showed an intenseimmunoreaction for MN.

Discussion

An adenoma-carcinoma morphological model has been proposed that involvesa sequence of histological changes from a normal colonic epitheliumthrough benign adenomas that become increasingly dysplastic and finallydevelop into cancer. [Faeron and Vogelstein, Cell, 61: 759-767 (1990).]Colorectal cancers typically develop over decades and appear to requireseveral genetic events for the completion of the malignant phenotype.[Lengauer et al., Nature, 386: 623-627 (1997); and Kinzler andVogelstein, Cell, 87: 159-170 (1996).] One important consequence ofthese genetic aberrations is that cell proliferation is abnormallyincreased in premalignant and malignant lesions of the colorectalepithelium. [Risio, M., J. Cell Biochem, 16G: 79-87 (1992).]

Several important aspects of MN expression emerged from thisinvestigation: 1) hyperplastic polyps showed immunoreaction in thecryptal area, as reported for normal mucosa, 2) MN was more widespreadand abundant in the surface epithelium of adenomas, 3) the stainingpattern was more diffuse in carcinomas than in benign lesions, 4) of allthe carcinoma samples, the most prominent reactions were observed in thetumors with a mucinous component, and 5) the desmoplastic connectivetissue of some malignant lesions revealed an intense immunoreaction.Furthermore, the results confirmed that MN is expressed on thebasolateral membranes of enterocytes in hyperplastic polyps andadenomas, whereas it was the polarized expression of MN on cell surfacesthat was affected in carcinomas.

To demonstrate more precisely the association of MN expression withproliferation, immunohistochemistry for Ki-67, which is one of the mostreliable markers of cell proliferation was used. Ki-67 and MN wereco-expressed in the same area in these lesions, indicating that MNexpression is indeed related to cell proliferation.

In summary, the present results show that the majority of colorectaltumors display abnormal expression of MN, pointing to MN's involvementin their pathogenesis. Furthermore, the co-occurrence of MN and Ki-67 atthe site of rapid cell proliferation indicates that MN could be used asa biomarker of increased cell proliferation in the colorectal mucosa.High expression of MN in premalignant lesions such as adenomas suggestsMN's usefulness in early diagnosis of colorectal tumors.

EXAMPLE 8 Immunohistochemistry of MN in Human Gut Reveals PolarizedExpression in the Epithelial Cells with the Highest ProliferativeCapacity

Saarnio et al., J. Histochem. Cytochem, 46(4): 497-509 (1998), presentsthe localization of MN (MN/CA IX) in the human gut and compares itsdistribution to those of carbonic anhydrases (CAs) I, II and IV, whichare known to be expressed in intestinal epithelium. Immunohistochemicalstaining of the human gut with the M75 MAb revealed prominent polarizedstaining for MN in the basolateral surfaces of the enterocytes ofduodenum and jejunum, the reaction being most intense in the crypts. Amoderate reaction was also seen in the crypts of ileal mucosa, whereasthe staining became generally weaker in the large intestine.

The results of the study indicated isozyme-specific regulation of MNexpression along the cranial-caudal axis of the human gut and place MNprotein at the sites of rapid cell proliferation. The uniquelocalization of MN on the basolateral surfaces of proliferating cryptenterocytes indicates that it may serve as a ligand or a receptor foranother protein that regulates intercellular communication or cellproliferation. Further, MN has a completely conserved active site domainof CAs suggesting that it could also participate in CO₂/bicarbonatehomeostasis.

Immunohistochemistry of MN in the Gut

MN showed a unique staining pattern in human gut. First, it was highlyexpressed in duodenum and jejunum (as in stomach) and was distinctlyless expressed in more distal segments of the gut. Second, theintestinal staining for MN was most intense in the crypts. Third, thepositive signal for MN was confined to the basolateral plasma membranesin all stained enterocytes.

Table 6 shows the immunohistochemical localization of MN in differentsegments of the gut. Duodenum and jejunum show intense staining in thecrypt enterocytes, whereas the epithelial cells in upper portions andtips of the villi showed only a weak immunoreaction. MN was also presentin enterocytes of human ileum, although the positive reaction was muchweaker than in duodenum and jejunum. In the large intestine, thebasolateral surfaces of the crypt enterocytes showed positive stainingfor MN, with a gradual change in staining intensity in a distaldirection. The reaction was still moderate in cecum and ascending colon,whereas the transverse and descending parts showed considerably weakersignals. Only sporadic enterocytes in sigmoid colon and rectum exhibitedweak basolateral staining. TABLE 7 Distribution of MN/CA IX, CA I, CAII, and CA IV in Human Gut* Histological MN/CA Segment site IX CA I CAII CA IV Small intestine Duodenum SE +/− − +++ + BC +++ − ++ − BR − − ++− ENDO − +++ − +++ Jejunum SE +/− − +++ +/− BC +++ + − − ENDO − +++ −+++ Ileum SE +/− − +/− +/− BC ++ + − − ENDO − +++ − +++ Large intestineCecum SE − +++ +++ +++ BG ++ − − − ENDO − +++ − +++ Ascending colon SE −+++ +++ +++ BG ++ − − − ENDO − +++ − +++ Transverse colon SE − +++ ++++++ BG + − − − ENDO − +++ − +++ Descending colon SE − +++ +++ +++ BG + −− − ENDO − +++ − +++ Sigmoid colon SE − +++ +++ +++ BG +/− − − − ENDO −+++ − +++ Rectum SE − +++ +++ +++ BG +/− − − − ENDO − +++ − +++*BC, base of the crypts; BR, Brunner's glands; BG, base of the glands;ENDO, submucosal capillary endothelium; SE, surface epithelium; −, nostaining; +/−, sporadic stained cells; +, weak staining; ++, moderatestaining; +++, intense staining.

The major aim of the study was to investigate the regional, cellular andsubcellular localization of MN in the human gut. The availability ofother purified CAs expressed in the gut and antibody reagents to each ofthem allowed confirmation of the specificity of the anti-MN antibody(M75 MAb), and to compare MN's sites of expression to those of other CAsin parallel tissue sections. Another opportunity to verify thespecificity of the M75 immunostainings was provided by the expression ofMN in COS-7 cells, which produced immunoreactive protein that waslocalized to the plasma membrane. As reported for Western blots of humanstomach, twin proteins identified by the M75 MAb on Western blots oftransfected COS-7 cells were the 58-kDa form and 54-kDa form, the latterbeing barely apparent in this study. The M75 MAb did not cross-reactwith purified CA I, II, IV and VI, all of which are expressed in variousparts of the alimentary tract [Parkkila and Parkkila, Scand. J.Gastorenteroli, 31: 305 (1996)], providing further evidence that theimmunostaining for MN in the gut is isozyme-specific.

Saarnio et al. demonstrated that the distribution of MN in the gut hasunique features. First, its subcellular localization is restricted tothe basolateral surfaces of epithelial cells. Second, its cellulardistribution is restricted to the crypt enterocytes, which is not seenwith any other CA. Third, its regional expression is distinctivecompared to other CAs, being most intense in duodenum and jejunum, anddecreasing distally from moderate expression in crypts of ileum, cecumand ascending colon to only weak and sporadic expression in distal largeintestine.

Restriction of MN to the epithelial cells with the greatestproliferative capacity is consistent with MN's proposed role in cellproliferation. Stem cells located in the crypts are a source for thecontinuous and rapid renewal of the epithelium [Gordon, J. I., J. Cell.Biol., 108: 1187-1194 (1989)]. Earlier studies in mice have shown thateach small intestinal crypt produces an average of 13-16 new cells/hour[Gordon et al., FASEB J., 6: 3039-3050 (1992)]. Cell differentiation andcell migration are coupled events in the gut. The epithelial cellsdifferentiate and mature during migration from the crypt to the apex ofthe villus or surface epithelial cuff [Gordon, J. I., supra (1989);Gordon et al., supra (1992) and Lipkin, M., Ann. Rev. Physiol., 47:175-197 (1985)]. This process is completed in a few days, which makesthe mammalian intestinal epithelium a unique model system for studyingcell differentiation. That MN is much more abundant in the proliferatingcryptal epithelium than in the upper part of the mucosa, indicates thatit could be a useful marker to study the proliferation anddifferentiation of the intestinal epithelium. Further support for itsrole as a marker for cell proliferation is obtained from studies showingthat the rate of cell proliferation is highest in the proximal segmentsof the intestine [Lipkin, M., Annu. Rev. Physiol., 47: 175 (1985);Gordon, J. I., supra (1989)], which was also the site of the highest MNexpression.

As a CA-homologous transmembrane protein, MN has some structuralsimilarity with recently described receptor-type protein tyrosinephosphatases (RPTPs) β and γ, which are members of a distinct group ofphosphatases that have CA-homologous regions in their extracellulardomains [Krueger and Saito, PNAS (USA) 89: 7417-7421 (1992); Levy etal., J. Biol. Chem., 268: 10573-10581 (1993); and Barnea et al., Mol.Cell. Biol., 13: 1497-1505 (1993)]. Both transmembrane and secretoryforms of RPTPβ have been found to be identical with a chondroitinsulfate proteoglycan called phosphacan [Barnea et al., Cell, 76: 205(1994); Maurel et al., PNAS (USA), 91: 2512-2516 (1994); Shitara et al.,J. Biol. Chem., 269: 20189-20193 (1994); and Barnea et al., J. Biol.Chem., 269: 14349-14352 (1994)].

The CA-like domains of the RPTPβ and -γ showed about 30-50% amino acididentity with CAs [Krueger and Saito, supra (1992); Barnea et al., supra(1993); and Levy et al., supra (1993). Unlike MN, RPTPβ and -γ have onlyone of the three conserved histidine residues required to bind to zincion in catalytically active CAs. Therefore, it is unlikely that theyhave any CA activity.

Recent studies have indicated that the CA-like domain of RPTPβ binds tocontactin, a neuronal cell recognition molecule [Peles et al., Cell, 82:251-260 (1995)], and to tenascin, an extracellular matrix protein [Milevet al., J Biol Chem. 270: 24650-24653 (1995)]. The CA-like domaininduced cell adhesion and neurite growth of primary tectal neurons, anddifferentiation of neuroblastoma cells [Peles et al., supra (1995)]. MNexpression, being regulated by cell density and being greatest inbasolateral surfaces of proliferating cryptal enterocytes, indicatesthat MN may also serve as a ligand or a receptor for another proteinthat regulates intercellular communication and cell proliferation.However, unlike the RPTPβ and -γ, MN has a completely conserved activesite domain that suggests that it could also participate in carbondioxide/bicarbonate homeostasis.

EXAMPLE 9 Accessibility In Vivo of MN Protein Expressed in Tumor Cellsand in Stomach

Lewis rats (384 g) carrying a BP6 subcutaneous tumor (about 1 cm indiameter) expressing rat MN protein were injected intraperitoneally(i.p.) with ¹²⁵I-M75 Mab (2.5×10⁶ cpm). Five days later, 0.5-1 g piecesof the tumor and organs were weighed and their radioactivity wasmeasured by a gamma counter.

Table 8 summarizes the results. The highest radioactivity was present inthe tumor. Relatively high radioactivity was found in the liver andkidney, apparently reflecting the clearance of mouse IgG from the blood.The stomach continued a relatively low level of radioactivity,indicating that the M75 Mab had only limited access to MN proteinexposed in the gastric mucosa. TABLE 8 Distribution of radioactivity of¹²⁵I-M75 in rat organs and in the tumor Organ cpm/g Kidney 2153 2184Spleen 653 555 Liver 1993 1880 Lung 1183 1025 Blood 1449 Heart 568 477Stomach 1184 1170 Testis 812 779 Tail 647 Tumor 3646 4058 3333 8653 3839

EXAMPLE 10 FACS Analysis of MN Protein Expression in CGL3Cells—Apoptosis

A FACS investigation was designed to determine the conditions thatinfluence the synthesis of MN protein and to analyse the cell cycledistribution of MN-positive versus MN-negative cells in a CGL3population stimulated to apoptosis. Previous Western blotting analyseshave shown CGL3 cells to express a relatively high amount of MN proteinunder different cultivation conditions. CGL3 cells are considered aconstitutive producer of MN proteins. However, Western blotting does notrecognize small differences in the level of protein. In contrast FACSallows the detection of individual MN-positive cells, a calculation oftheir percentage in the analysed population, an estimation of the levelof MN protein in the cells, and a determination of the cell cycledistribution.

To study the effect of cultivation conditions on MN expression in CGL3cells, the CGL3 cells were plated in different relative densities andserum concentrations. Three days after plating, the cells werecollected, surface labeled by M75 Mab followed by FITC-conjugatedanti-mouse IgG and immediately analysed by FACS.

The analysis showed that in adherent cells, MN expression is dependenton cell density as is HeLa cells. However, low density cultures stillproduced detectable amounts of MN protein. In low density cultures,serum concentration does not seem to play a role. In relatively highdensity cultures, a decreasing serum concentration resulted in slightlydiminished MN expression, probably due to a lower density that the cellswere able to reach during the three days of cultivation.

The effect of the actual cell density is remarkable, and MN expression(detectable in 15-90% of the cells) represents a very sensitivemonitoring factor. In all experiments, there was about a 5% higherpercentage of cycling cells in the MN-positive part of the population,compared to the MN-negative part. That fact prompted the analysis of thecell cycle distribution of MN-positive CGL3 cells under unfavorablegrowth conditions, that is, after induction of apoptosis.

Apoptosis

CGL3 cells were stimulated to apoptotic death by several drugs,including cycloheximide, actimonycin D and dexamethasone. The FACS studyshowed that the onset of apoptosis is delayed in MN-positive cellssuggesting a protective role of MN in this process. It was also observedthat the induction of apoptosis resulted in the down-regulation of MNexpression in a time-dependent manner. That same phenomenon wasdescribed for Bcl-2 anti-apoptotic protein, and there is existingopinion that the down-regulation of certain regulatory genes duringapoptosis sensitizes the cells to undergo apoptotic death. To prove therole of MN in apoptosis, a similar study with cells transfected by MNcDNA is to be performed.

The preliminary results indicate the possible involvement of MN in thesuppression of apoptosis. The recent view that tumors arise both as aconsequence of increased proliferation and decreased cell death appearsto be consistent with the association of the MN protein with tumors invivo.

ATCC Deposits. The materials listed below were deposited with theAmerican Type Culture Collection (ATCC) now at 10810 University Blvd.,Manassus, Va. 20110-2209 (USA). The deposits were made under theprovisions of the Budapest Treaty on the International Recognition ofDeposited Microorganisms for the Purposes of Patent Procedure andRegulations thereunder (Budapest Treaty). Maintenance of a viableculture is assured for thirty years from the date of deposit. Thehybridomas and plasmids will be made available by the ATCC under theterms of the Budapest Treaty, and subject to an agreement between theApplicants and the ATCC which assures unrestricted availability of thedeposited hybridomas and plasmids to the public upon the granting ofpatent from the instant application. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anyGovernment in accordance with its patent laws. Hybridoma Deposit DateATCC # VU-M75 Sep. 17, 1992 HB 11128 MN 12.2.2 Jun. 9, 1994 HB 11647

Plasmid Deposit Date ATCC # A4a Jun. 6, 1995 97199 XE1 Jun. 6, 199597200 XE3 Jun. 6, 1995 97198

The description of the foregoing embodiments of the invention have beenpresented for purposes of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to explain the principles of the invention and itspractical application to enable thereby others skilled in the art toutilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated.

All references cited herein are hereby incorporated by reference.

1-23. (canceled)
 24. An anti-anti-idiotype antibody to an anti-idiotypeantibody to a MN-specific antibody, wherein said anti-idiotype antibodycomprises an internal image corresponding to an epitope of MN protein,wherein said MN protein has an amino acid sequence of SEQ ID NO: 2 thatis encoded by a nucleic acid selected from the group consisting of: (a)SEQ ID NO: 1; and (b) polynucleotides that differ from SEQ ID NO: 1 dueto the degeneracy of the genetic code.
 25. An anti-anti-idiotypeantibody according to claim 24 wherein said MN-specific antibody iseither the M75 monoclonal antibody secreted from the hybridoma VU-M75,which was deposited at the American Type Culture Collection under ATCCNo. HB 11128, or the MN12 monoclonal antibody that is secreted from thehybridoma MN 12.2.2, which was deposited at the American Type CultureCollection under ATCC No. HB
 11647. 26. An anti-anti-idiotype antibodyaccording to claim 24 which is monoclonal.
 27. An anti-anti-idiotypeantibody according to claim 24 which is polyclonal. 28-29. (canceled)30. The anti-anti-idiotype antibody according to claim 24 which isrecombinantly produced.
 31. The anti-anti-idiotype antibody according toclaim 24 which is an antigen-binding antibody fragment.
 32. A method oftreating a patient with a preneoplastic and/or neoplastic disease, thatis characterized by the abnormal expression of MN protein, byadministering to said patient a therapeutically effective amount of ananti-anti-idiotype MN-specific antibody.
 33. The method according toclaim 32, wherein said anti-anti-idiotype antibody is monoclonal. 34.The method according to claim 32, wherein said anti-anti-idiotypeantibody is polyclonal.
 35. The method according to claim 34, whereinsaid polyclonal anti-anti-idiotype antibody is administered in a serum.36. The method according to claim 32 further comprising administering tosaid patient a therapeutically effective amount of one or morecytokines.
 37. The method according to claim 36 wherein said cytokine iseither interferon or interleukin-2, or said cytokines are interferon andinterleukin-2.